Figure 2 . ( B ) A small vesicle generation assay as above was performed with the indicated conditions . Slowly - sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction . 3 - MA , 5 mM ; Wortmannin , 20 nM ; S , starved Atg5 KO MEF cytosol ; N , untreated Atg5 KO MEF cytosol ; − / AP , in the absence of ATP regeneration and GTP and in the presence of apyrase ( AP ) . ( D ) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K , P and F1 - 4 fractions ( upper four panels : Small vesicle ) . SDS - PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction ( lower five panels : Fraction input ) . TFR , transferrin receptor ( E ) A small vesicle generation assay as shown in ( A ) was performed in the presence of indicated proteins or drugs . Slowly - sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST , H79G ( Sar1A ( H79G ) , 0 . 7 μM ) , wortmannin ( 20 nM ) , or BFA ( brefeldin A , 0 . 5 μg / ml ) . ( F ) A small vesicle generation assay as shown in ( A ) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A ( H79G ) or 3 - MA followed by a lipidation reaction . ( G ) Atg5 KO MEFs were starved for 30 min . Differential centrifugation as shown in Figure 1D was performed to collect the 25K × g and 100K × g pellet fractions . The LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 ( H79G ) . Quantification of lipidation activity is shown as the ratio of LC3 - II to LC3 - I ( II / I ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.(B) A small vesicle generation assay as above was performed with the indicated conditions. Slowly-sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction. 3-MA, 5 mM; Wortmannin, 20 nM; S, starved Atg5 KO MEF cytosol; N, untreated Atg5 KO MEF cytosol; −/AP, in the absence of ATP regeneration and GTP and in the presence of apyrase (AP).(D) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K, P and F1-4 fractions (upper four panels: Small vesicle). SDS-PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction (lower five panels: Fraction input). TFR, transferrin receptor(E) A small vesicle generation assay as shown in (A) was performed in the presence of indicated proteins or drugs. Slowly-sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST, H79G (Sar1A (H79G), 0.7 μM), wortmannin (20 nM), or BFA (brefeldin A, 0.5 μg/ml).(F) A small vesicle generation assay as shown in (A) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A (H79G) or 3-MA followed by a lipidation reaction.(G) Atg5 KO MEFs were starved for 30 min. Differential centrifugation as shown in Figure 1D was performed to collect the 25K ×g and 100K ×g pellet fractions. The LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 (H79G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).


Figure 3 . ( A ) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min . Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 ( F2 in Figure 2C , D ) of the OptiPrep gradient in the three - step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes . ( B ) Atg5 KO MEFs were transfected with a plasmid encoding an HA - tagged SEC31A . After transfection ( 24 hr ) , the cells were treated as shown in ( A ) . Immunofluorescence was performed with anti - ERGIC53 and anti - HA antibodies and the cells were examined by confocal microscopy . Bar : 10 μM ( C ) Quantification of SEC31A overlapped with ERGIC53 shown in ( B ) using the Pearson ' s correlation coefficient ( PCC ) . Error bars represent standard deviations from ∼ 20 cells in three independent experiments . p values were calculated by T - test .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.(A) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min. Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 (F2 in Figure 2C,D) of the OptiPrep gradient in the three-step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes.(B) Atg5 KO MEFs were transfected with a plasmid encoding an HA-tagged SEC31A. After transfection (24 hr), the cells were treated as shown in (A). Immunofluorescence was performed with anti-ERGIC53 and anti-HA antibodies and the cells were examined by confocal microscopy. Bar: 10 μM (C) Quantification of SEC31A overlapped with ERGIC53 shown in (B) using the Pearson's correlation coefficient (PCC). Error bars represent standard deviations from ∼20 cells in three independent experiments. p values were calculated by T-test.


Figure 1A - D ) Confocal image analysis of cryostat sections from adult CX3CR1 + / GFP ; Sfrp1 + / βgal ( A , B ) ; Sfrp1 + / βgal ( C ) and CX3CR1 + / GFP ; Sfrp1 - / - ( D ) and mouse brains three days after intra - cortical infusion of saline or LPS . Sections were immunostained for βgal ( magenta , green in C ) and Iba1 ( green in A ; red in C ) or Sfrp1 ( magenta ) and GFP ( green , B and D ) , and GFAP ( cyan in A ; B , D , red in C ) . Arrowheads indicate βgal / GFAP ( A ) and Sfrp1 / GFP co - localization ( B ) . No Sfrp1 protein is detected in the null mice independently of the treatment ( two bottom lines ) . Scale bar 25μm . E ) ELISA determination of Sfrp1 levels in brain extracts from 3 months - old wt and Sfrp1 - / - mice . WT mice were injected either with saline or LPS . Three days after injection the region around the injected side ( 10 mm3 cortical cube ) was isolated and SFRP1 content compared with that present in similar region of non - injected or Sfrp1 - / - mice used as negative control ( n = 5 mice for each group ) . Error bars represent Standard Error . Statistical significance : ns P > 0 . 5 * * * * P < 0 . 0001 ; One - way ANOVA followed by Bonferroni multiple comparisons test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-D) Confocal image analysis of cryostat sections from adult CX3CR1+/GFP;Sfrp1+/βgal (A,B); Sfrp1+/βgal (C) and CX3CR1+/GFP;Sfrp1-/- (D) and mouse brains three days after intra-cortical infusion of saline or LPS. Sections were immunostained for βgal (magenta, green in C) and Iba1 (green in A; red in C) or Sfrp1 (magenta) and GFP (green, B and D), and GFAP (cyan in A;B, D, red in C). Arrowheads indicate βgal/GFAP (A) and Sfrp1/GFP co-localization (B). No Sfrp1 protein is detected in the null mice independently of the treatment (two bottom lines). Scale bar 25μm. E) ELISA determination of Sfrp1 levels in brain extracts from 3 months-old wt and Sfrp1-/- mice. WT mice were injected either with saline or LPS. Three days after injection the region around the injected side (10 mm3 cortical cube) was isolated and SFRP1 content compared with that present in similar region of non-injected or Sfrp1-/- mice used as negative control (n=5 mice for each group). Error bars represent Standard Error. Statistical significance: ns P>0.5 ****P<0.0001; One-way ANOVA followed by Bonferroni multiple comparisons test.


Figure 2A ) Coronal cryostat sections of LV - IRES - Gfp or LV - Sfrp1 - IRES - Gfp infected brains 1 - month post - infusion , immunostained for SFRP1 ( magenta ) Iba1 ( green ) GFAP ( cyan ) , or TauP ( red ) . Lv , lateral ventricle . Scale bar 100μm . B ) The graph shows the normalised level of GFAP , Iba1 and CD45 immunoreactivity ( IR ) and the area occupied by TauP signal ( n = 24 acquisitions , white dots ; N = 3 mice , black dots , for each group ) , normalized to LV - IRES - Gfp infected brains . Error bars represent Standard Error . Statistical significance : * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 by two - sided Student ' s t - test . C , D ) Coronal sections from wt and Sfrp1 - / - mouse brains three days after infusion of saline or LPS , immunostained for GFAP ( cyan , C ) or CD45 ( magenta , D ) . The images are high power views of the somatosensory cortex ( lower power view in Fig EV1D ) . Scale bar 60μm . E , F ) The graphs show the normalised levels of immunoreactivity ( IR ) for GFAP ( E ) and CD45 ( F , P = 0 , 006 ) present in cortical sections ( n = 24 acquisitions white dots ; from N = 3 animals , black dots , per group ) from wt and Sfrp1 - / - animals infused with saline or LPS . Bars represent Standard Error . Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black . * * or # # P < 0 . 01 ; * * * or # # # P < 0 . 001 ; * * * * or # # # # P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons test . * and # indicate significance between genotypes and treatments , respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) Coronal cryostat sections of LV-IRES-Gfp or LV-Sfrp1-IRES-Gfp infected brains 1-month post-infusion, immunostained for SFRP1 (magenta) Iba1 (green) GFAP (cyan), or TauP (red). Lv, lateral ventricle. Scale bar 100μm. B) The graph shows the normalised level of GFAP, Iba1 and CD45 immunoreactivity (IR) and the area occupied by TauP signal (n=24 acquisitions, white dots; N=3 mice, black dots, for each group), normalized to LV-IRES-Gfp infected brains. Error bars represent Standard Error. Statistical significance: *P<0.05; **P<0.01; ****P<0.0001 by two-sided Student's t-test. C, D) Coronal sections from wt and Sfrp1-/- mouse brains three days after infusion of saline or LPS, immunostained for GFAP (cyan, C) or CD45 (magenta, D). The images are high power views of the somatosensory cortex (lower power view in Fig EV1D). Scale bar 60μm. E, F) The graphs show the normalised levels of immunoreactivity (IR) for GFAP (E) and CD45 (F, P=0,006) present in cortical sections (n=24 acquisitions white dots; from N=3 animals, black dots, per group) from wt and Sfrp1-/- animals infused with saline or LPS. Bars represent Standard Error. Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black. ** or ## P<0.01; *** or ### P<0.001; **** or #### P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test. * and # indicate significance between genotypes and treatments, respectively.


Figure 3C ) Wt and Sfrp1 - / - mice immunized with MOG and sacrificed 16 days post immunization . Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4 , Iba1 , GFAP and MBP . Images show the region dorsal fasciculus . White arrowheads point to the disruption of the pia surface in wt . Scale bar 100μm . D ) Quantification of CD4 + infiltrated lymphocytes , Iba1 normalised immunoreactivity , MBP normalised immunoreactivity and MBP immune - reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1 - / - mice 16 days after immunization ( n = 16 acquisitions , white dots ; from N = 4 animals , black dots , per genotype ) . Error bars represent Standard Error . Statistic refers to number of acquisitions .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C) Wt and Sfrp1-/- mice immunized with MOG and sacrificed 16 days post immunization. Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4, Iba1, GFAP and MBP. Images show the region dorsal fasciculus. White arrowheads point to the disruption of the pia surface in wt. Scale bar 100μm. D) Quantification of CD4+ infiltrated lymphocytes, Iba1 normalised immunoreactivity, MBP normalised immunoreactivity and MBP immune-reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1-/- mice 16 days after immunization (n=16 acquisitions, white dots; from N=4 animals, black dots, per genotype). Error bars represent Standard Error. Statistic refers to number of acquisitions.


Figure 4D ) ELISA determination of SFRP1 levels present in the media of microglia ( n = 4 cultures per genotype ) , astrocytes ( n = 4 cultures per genotype ) or mixed astrocytes and microglial ( 2 : 1 , n = 7 cultures per genotype ) cultures derived from wt or Sfrp1 - / - pups exposed for 24h to saline or LPS ( 1μg / ml ) . Error bars represent Standard Error . Statistical significance : * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; One - way ANOVA followed by Bonferroni Test . Secretory profile of cytokines present in the medium of microglia , astrocytes or mixed astrocytes and microglial ( 2 : 1 ) culture as above ( n = 5 - 6 cultures type - microglia , astrocytes or mixed cultures - and genotype ) . The content of IL1β , IL10 was determined by Multiplex ELISA . Values are represented in absolute concentration . Error bars represent Standard Error . Statistical significance : ns P > 0 . 05 ; * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons test . F ) Secretory profile of cytokines present in the medium of microglia , astrocytes or mixed astrocytes and microglial ( 2 : 1 ) culture as above ( n = 5 - 6 cultures type - microglia , astrocytes or mixed cultures - and genotype ) . The content of IFNγ , TNFα , IL4 , IL6 was determined by Multiplex ELISA . Values are represented in absolute concentration . Error bars represent Standard Error . Statistical significance : ns P > 0 . 05 ; * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4D) ELISA determination of SFRP1 levels present in the media of microglia (n=4 cultures per genotype), astrocytes (n=4 cultures per genotype) or mixed astrocytes and microglial (2:1, n=7 cultures per genotype) cultures derived from wt or Sfrp1-/- pups exposed for 24h to saline or LPS (1μg/ml). Error bars represent Standard Error. Statistical significance: *P<0.05; ***P<0.001; ****P<0.0001; One-way ANOVA followed by Bonferroni Test.Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IL1β, IL10 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.F) Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IFNγ, TNFα, IL4, IL6 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.


Figure 5B , C ) Volcano plots of differential gene expression from CX3CR1GFP / + ( B ) or CX3CR1GFP / + ; Sfrp1 - / - ( C ) microglial cell in response to LPS . Data are represented as Log2 Fold Change vs - Log10 adjusted p - value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method . Blue vertical lines represent an increase of 75 and 400 % in the expression levels respectively . Green horizontal lines represent a 0 . 05 or 0 . 01 adjusted p - value of statistical significance respectively . Genes with an expression change higher than 75 % and an adjusted p - value lower than 0 . 05 are highlighted in dark red ( + FC ) and dark blue ( - FC ) . Genes with an expression change higher than 400 % and an adjusted p - value lower than 0 . 01 are coloured in light red ( + FC ) and light blue ( - FC ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B, C) Volcano plots of differential gene expression from CX3CR1GFP/+ (B) or CX3CR1GFP/+;Sfrp1-/- (C) microglial cell in response to LPS. Data are represented as Log2 Fold Change vs -Log10 adjusted p-value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method. Blue vertical lines represent an increase of 75 and 400% in the expression levels respectively. Green horizontal lines represent a 0.05 or 0.01 adjusted p-value of statistical significance respectively. Genes with an expression change higher than 75% and an adjusted p-value lower than 0.05 are highlighted in dark red (+FC) and dark blue (-FC). Genes with an expression change higher than 400% and an adjusted p-value lower than 0.01 are coloured in light red (+FC) and light blue (-FC).


Figure 6B ) Integrative Genomics Viewer transcription profile of represented genes in microglial cells isolated for saline and LPS treated CX3CR1GFP / + and CX3CR1GFP / + ; Sfrp1 - / - mouse brains . Scale bar : 2 Kbp .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B) Integrative Genomics Viewer transcription profile of represented genes in microglial cells isolated for saline and LPS treated CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- mouse brains. Scale bar: 2 Kbp.


Figure 1A . The localization of NSUN3 was analysed in HEK293 cells stably expressing NSUN3 - GFP ( green ) . NSUN3 - GFP and staining with a Mitotracker ( red ) are shown separately and in an overlay with DAPI to indicate nuclei . The scale bar represents 5 m . B . To analyse submitochondrial localization of NSUN3 , human mitochondria were isolated and either left untreated , swollen in hypotonic buffer ( Mitoplasts ) or solubilized by sonication ( Sonic . ) before treatment with different amounts of Proteinase K ( PK ) where indicated , followed by SDS - PAGE and Western blotting using antibodies against human TIM44 , TIM23 , TOM70 or FLAG - tagged NSUN3 . Note that TIM44 localises to the matrix , while the N - terminus of TIM23 localises to the intermembrane space and TOM70 is largely exposed on the mitochondrial surface . The asterisk indicates a cross - reaction of the TOM70 antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. The localization of NSUN3 was analysed in HEK293 cells stably expressing NSUN3-GFP (green). NSUN3-GFP and staining with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.B. To analyse submitochondrial localization of NSUN3, human mitochondria were isolated and either left untreated, swollen in hypotonic buffer (Mitoplasts) or solubilized by sonication (Sonic.) before treatment with different amounts of Proteinase K (PK) where indicated, followed by SDS-PAGE and Western blotting using antibodies against human TIM44, TIM23, TOM70 or FLAG-tagged NSUN3. Note that TIM44 localises to the matrix, while the N-terminus of TIM23 localises to the intermembrane space and TOM70 is largely exposed on the mitochondrial surface. The asterisk indicates a cross-reaction of the TOM70 antibody.


Figure 2B . HEK293 cells expressing NSUN3 - HisPrcFLAG ( NSUN3 ) or the HisPrcFLAG - tag alone ( FLAG ) were either not crosslinked ( - ) , UV crosslinked ( UV ) or treated with 5 - azacytidine ( 5 - AzaC ) . The protein - RNA complexes were affinity purified and the bound RNA was trimmed , end - labelled with 32P phosphate and ligated to linkers . Protein - RNA complexes were separated by SDS - PAGE , transferred to nitrocellulose and exposed to an X - ray film . G . 5 - AzaC crosslinking was performed and RNA associated with wildtype NSUN3 , the catalytically inactive NSUN3 mutant ( C265A ) or the FLAG tag alone was isolated as described in ( B ) . The RNA analysed by northern blot using probes against the mt - tRNAMet , mt - tRNAPro and mt - tRNAGlu . Inputs ( 0 . 1 % ) are shown on the left and eluates ( 50 % ) on the right .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. HEK293 cells expressing NSUN3-HisPrcFLAG (NSUN3) or the HisPrcFLAG-tag alone (FLAG) were either not crosslinked (-), UV crosslinked (UV) or treated with 5-azacytidine (5-AzaC). The protein-RNA complexes were affinity purified and the bound RNA was trimmed, end-labelled with 32P phosphate and ligated to linkers. Protein-RNA complexes were separated by SDS-PAGE, transferred to nitrocellulose and exposed to an X-ray film.G. 5-AzaC crosslinking was performed and RNA associated with wildtype NSUN3, the catalytically inactive NSUN3 mutant (C265A) or the FLAG tag alone was isolated as described in (B). The RNA analysed by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.


Figure 3A . In vitro methylation reactions were performed using recombinant His14 - MBP - NSUN3 ( NSUN3 ) or the catalytically inactive mutant His14 - MBP - NSUN3 - C265A ( C265A ) , 3H - labelled S - adenosyl methionine as a methyl group donor and in vitro transcribed mt - tRNAMet , mt - tRNAPro and mt - tRNAGlu . The RNA was then separated on a denaturing polyacrylamide gel , stained with ethidium bromide ( EtBr ) to indicate inputs and exposed to an X - ray film to analyse methylation ( 3H - Me ) . B . The distribution of Illumina sequence reads along the mt - tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV ( light grey ) or 5 - AzaC crosslinking ( dark grey ) is given as reads per million mapped reads . The position of the anticodon is indicated by a bar . D . In vitro methylation assays were performed as described in ( A ) with His14 - MBP - NSUN3 and in vitro transcribed wildtype mt - tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in ( C ) . Two exposure times of X - ray films are shown : 16 hours ( short ) , 3 days ( long ) . E . In vitro methylation assay of in vitro transcribed mt - tRNAMet and chemically synthesized mt - tRNAMet containing an m5C modification at the wobble position . The experiment and analysis were performed as described in ( A ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. In vitro methylation reactions were performed using recombinant His14-MBP-NSUN3 (NSUN3) or the catalytically inactive mutant His14-MBP-NSUN3-C265A (C265A), 3H-labelled S-adenosyl methionine as a methyl group donor and in vitro transcribed mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. The RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr) to indicate inputs and exposed to an X-ray film to analyse methylation (3H-Me).B. The distribution of Illumina sequence reads along the mt-tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV (light grey) or 5-AzaC crosslinking (dark grey) is given as reads per million mapped reads. The position of the anticodon is indicated by a bar.D. In vitro methylation assays were performed as described in (A) with His14-MBP-NSUN3 and in vitro transcribed wildtype mt-tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in (C). Two exposure times of X-ray films are shown: 16 hours (short), 3 days (long).E. In vitro methylation assay of in vitro transcribed mt-tRNAMet and chemically synthesized mt-tRNAMet containing an m5C modification at the wobble position. The experiment and analysis were performed as described in (A).


Figure 4B . In vitro methylation assays using [ 3H - methyl ] - labelled S - adenosyl methionine , the in vitro transcripts of the mt - tRNAMet mutants described in ( A ) and recombinant His14 - MBP - NSUN3 . RNA was then separated on a denaturing polyacrylamide gel , stained with ethidium bromide ( EtBr ) , dried and exposed to an X - ray film to detect methylated transcripts ( 3H - Me ) . C . In vitro methylation assay using chemically synthesised ASL . The experiment and analysis were performed as described in ( B ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. In vitro methylation assays using [3H-methyl]-labelled S-adenosyl methionine, the in vitro transcripts of the mt-tRNAMet mutants described in (A) and recombinant His14-MBP-NSUN3. RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr), dried and exposed to an X-ray film to detect methylated transcripts (3H-Me).C. In vitro methylation assay using chemically synthesised ASL. The experiment and analysis were performed as described in (B).


Figure 5A . The localization of ABH1 was analysed by immunofluorescence in HEK293 cells . ABH1 ( green ) localisation and mitochondria stained with a Mitotracker ( red ) are shown separately and in an overlay with DAPI to indicate nuclei . The scale bar represents 5 m . B . HEK293 cells expressing ABH1 - HisPrcFLAG ( ABH1 ) or the HisPrcFLAG - tag alone ( FLAG ) were UV crosslinked ( UV ) and protein - RNA complexes were affinity purified . Co - precipitated RNA was isolated and analysis by northern blot using probes against the mt - tRNAMet , mt - tRNAPro and mt - tRNAGlu . Inputs ( 0 . 1 % ) are shown on the left and eluates ( 50 % ) on the right .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. The localization of ABH1 was analysed by immunofluorescence in HEK293 cells. ABH1 (green) localisation and mitochondria stained with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.B. HEK293 cells expressing ABH1-HisPrcFLAG (ABH1) or the HisPrcFLAG-tag alone (FLAG) were UV crosslinked (UV) and protein-RNA complexes were affinity purified. Co-precipitated RNA was isolated and analysis by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.


Figure 6A . In vitro transcribed mt - tRNAMet was methylated at C34 using recombinant NSUN3 and 3H - labelled S - adenosyl methionine as a methyl group donor . Radiolabelled mt - tRNAMet was re - extracted and then subjected to oxidation assays without protein ( - ) , with maltose binding protein ( MBP ) , with the dioxygenase FTO or using wildtype ( ABH1 ) or mutant ( R338A , D233A ) His14 - MBP - ABH1 . Besides ABH1 controls lacking a - ketoglutarate ( KG ) or Fe2 + ions , all samples contained a - ketoglutarate and Fe2 + ions . After oxidation , RNA was precipitated and tritium released upon oxidation of radiolabelled mt - tRNAMet was quantified in the supernatant and counts per minute ( CPM ) are shown for experiments performed in triplicate with error bars indicating ± SD ( upper panel ) . Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X - ray film to analyse the tritium retained ( 3H - Me ) . B . Synthetic anticodon stem loop ( ASL ) was radioactively labelled and subjected to oxidation assays that were performed and analysed as described in ( A ) using no protein ( - ) , MBP or wildtype His14 - MBP - ABH1 ( ABH1 ) . Experiments were performed in triplicate with error bars indicating ± SD . C . Anion exchange HPLC analysis was performed on synthetic m5C containing ASL ( 20nt ) before and after oxidation by ABH1 . The small shift in retention time indicates formation of f5C modified RNA . The ABH1 oxidation product was then treated with NaBH4 to generate hm5C - modified RNA . All three samples were analysed by ESI - MS and the molecular weight ( m . w . ) is indicated on the HPLC trace . Only the f5C - containing RNA was labelled efficiently with 1 - ethyl - 2 , 3 , 3 - trimethylindoleninium - 5 - sulfonate ( TMI ) . D . The different retention times of m5C , hm5C and f5C - modified ASL RNA were confirmed by co - injection of samples shown in ( C ) . HPLC was performed as in C .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. In vitro transcribed mt-tRNAMet was methylated at C34 using recombinant NSUN3 and 3H-labelled S-adenosyl methionine as a methyl group donor. Radiolabelled mt-tRNAMet was re-extracted and then subjected to oxidation assays without protein (-), with maltose binding protein (MBP), with the dioxygenase FTO or using wildtype (ABH1) or mutant (R338A, D233A) His14-MBP-ABH1. Besides ABH1 controls lacking a-ketoglutarate (KG) or Fe2+ ions, all samples contained a-ketoglutarate and Fe2+ ions. After oxidation, RNA was precipitated and tritium released upon oxidation of radiolabelled mt-tRNAMet was quantified in the supernatant and counts per minute (CPM) are shown for experiments performed in triplicate with error bars indicating ± SD (upper panel). Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X-ray film to analyse the tritium retained (3H-Me).B. Synthetic anticodon stem loop (ASL) was radioactively labelled and subjected to oxidation assays that were performed and analysed as described in (A) using no protein (-), MBP or wildtype His14-MBP-ABH1 (ABH1). Experiments were performed in triplicate with error bars indicating ± SD.C. Anion exchange HPLC analysis was performed on synthetic m5C containing ASL (20nt) before and after oxidation by ABH1. The small shift in retention time indicates formation of f5C modified RNA. The ABH1 oxidation product was then treated with NaBH4 to generate hm5C-modified RNA. All three samples were analysed by ESI-MS and the molecular weight (m.w.) is indicated on the HPLC trace. Only the f5C-containing RNA was labelled efficiently with 1-ethyl-2,3,3-trimethylindoleninium-5-sulfonate (TMI).D. The different retention times of m5C, hm5C and f5C-modified ASL RNA were confirmed by co-injection of samples shown in (C). HPLC was performed as in C.


Figure 7A . MTIF2 - dependent reading of initiation codons AUG , AUA or AUU in the P site of the ribosome by unmodified ( unmod . ) or C34 - modified [ 14C ] Met - tRNAMet . Binding was determined by nitrocellulose filtration and [ 14C ] Met - tRNAMet retrieved on the membrane was quantified by scintillation counting . Binding in the absence of ribosomes ( no 70S ) or mRNA ( no mRNA ) served as controls . Data from three independent experiments are presented with error bars indicating ± SEM . The statistical significance of the results was analysed by T - test and is indicated by the asterisks in the graph . B . TUFM - dependent recognition of A site codons during elongation . Data from three independent experiments are presented with error bars indicating ± SEM and statistical analysis as in ( A ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. MTIF2-dependent reading of initiation codons AUG, AUA or AUU in the P site of the ribosome by unmodified (unmod.) or C34-modified [14C]Met-tRNAMet. Binding was determined by nitrocellulose filtration and [14C]Met-tRNAMet retrieved on the membrane was quantified by scintillation counting. Binding in the absence of ribosomes (no 70S) or mRNA (no mRNA) served as controls. Data from three independent experiments are presented with error bars indicating ± SEM. The statistical significance of the results was analysed by T-test and is indicated by the asterisks in the graph.B. TUFM-dependent recognition of A site codons during elongation. Data from three independent experiments are presented with error bars indicating ± SEM and statistical analysis as in (A).


Figure 8A . HeLa cells were transfected with two different siRNAs against NSUN3 ( siNSUN3 _ 1 , siNSUN3 _ 2 ) , ABH1 ( siABH1 _ 1 , siABH1 _ 2 ) or with nontarget ( siNT ) siRNA and the knockdown efficiency was analysed by quantitative PCR . The relative abundance of the NSUN3 or ABH1 mRNA was normalized to GAPDH levels . Data are presented as mean ± SD . B . Chemically synthesised f5C modified mt - tRNAMet and total RNA from wild - type ( WT ) cells or those transfected with siRNAs as in ( A ) were treated with TMI to specifically label f5C residues . Primer extension , using a radiolabelled antisense primer , was performed under limited dNTP conditions . Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder and RNAs were detected using a phosphorimager . C . Primer extension reactions were performed on total RNA from cells transfected with siRNAs as described in B . Stops corresponding to position C34 in mt - tRNAMet were quantified in three independent experiments and results are shown graphically as mean ± SD . D . RNA from wild - type HeLa cells and cells treated with siRNAs against NSUN3 or ABH1 ( as in A ) was either first reduced with NaBH4 or directly treated with bisulfite . After deamination and desulfonation , mt - tRNAMetRNAs were reverse transcribed , amplified , cloned and sequenced . The proportions of thymine ( grey ) generated by bisulfite - conversion or non - converted cytosine ( black ) at position 34 of mt - tRNAMet are shown . Note that for sequences from non - reduced samples , thymine can originate from unmodified or f5C - containing mt - tRNAMet , while in reduced samples it originates from unmodified cytosine .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. HeLa cells were transfected with two different siRNAs against NSUN3 (siNSUN3_1, siNSUN3_2), ABH1 (siABH1_1, siABH1_2) or with nontarget (siNT) siRNA and the knockdown efficiency was analysed by quantitative PCR. The relative abundance of the NSUN3 or ABH1 mRNA was normalized to GAPDH levels. Data are presented as mean ± SD.B. Chemically synthesised f5C modified mt-tRNAMet and total RNA from wild-type (WT) cells or those transfected with siRNAs as in (A) were treated with TMI to specifically label f5C residues. Primer extension, using a radiolabelled antisense primer, was performed under limited dNTP conditions. Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder and RNAs were detected using a phosphorimager.C. Primer extension reactions were performed on total RNA from cells transfected with siRNAs as described in B. Stops corresponding to position C34 in mt-tRNAMet were quantified in three independent experiments and results are shown graphically as mean ± SD.D. RNA from wild-type HeLa cells and cells treated with siRNAs against NSUN3 or ABH1 (as in A) was either first reduced with NaBH4 or directly treated with bisulfite. After deamination and desulfonation, mt-tRNAMetRNAs were reverse transcribed, amplified, cloned and sequenced. The proportions of thymine (grey) generated by bisulfite-conversion or non-converted cytosine (black) at position 34 of mt-tRNAMet are shown. Note that for sequences from non-reduced samples, thymine can originate from unmodified or f5C-containing mt-tRNAMet, while in reduced samples it originates from unmodified cytosine.


Figure 9A . HeLa cells were treated with non - target siRNAs ( siNT ) or those targeting NSUN3 ( siNSUN3 _ 1 or siNSUN3 _ 2 ) or ABH1 ( siABH1 _ 1 or siABH1 _ 2 ) for 72 h before labelling of mitochondrial translation products with [ 35S ] methionine . Protein samples were separated by SDS - PAGE then transferred to a membrane . Labelled proteins were detected using a phosphorimager and the levels of tubulin were determined by western blotting using an antibody against the endogenous protein for normalisation . B . Mitochondrially - translated proteins that could be clearly detected were quantified in three independent experiments and the results are shown graphically as mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A. HeLa cells were treated with non-target siRNAs (siNT) or those targeting NSUN3 (siNSUN3_1 or siNSUN3_2) or ABH1 (siABH1_1 or siABH1_2) for 72 h before labelling of mitochondrial translation products with [35S]methionine. Protein samples were separated by SDS-PAGE then transferred to a membrane. Labelled proteins were detected using a phosphorimager and the levels of tubulin were determined by western blotting using an antibody against the endogenous protein for normalisation.B. Mitochondrially-translated proteins that could be clearly detected were quantified in three independent experiments and the results are shown graphically as mean ± SD.


Figure 1 The SnRK2 OST1 activates ABFs for expression of a luciferase reporter ( LUC ) in yeast . Luciferase activity is given on a Log10 scale as relative counts of light emission per second ( rcps ) normalized to the cell density . The OST1 - mediated induction factor is indicated above the columns . In the absence of ABFs , the LUC activity was 3 x 103 rcps . Combining the ABA signaling components ABF2 , OST1 , the PP2C ABI1 , and RCAR11 provides ABA - controlled LUC expression . The reporter is under the control of a minimal promoter that contains ABA - responsive cis elements ( ABREs ) . Core ABA signaling involves the RCAR receptor that inhibits the ABA coreceptor ( PP2C ) in the presence of ABA and allows the activation of the protein kinase SnRK2 . SnRK2 phosphorylates and activates the key transcription factor ABF , which targets promoters containing ABRE . Reporter activity was determined 16 h after exogenous ABA ( 0 . 1 mM ) administration and referred to samples expressing ABF2 alone .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 The SnRK2 OST1 activates ABFs for expression of a luciferase reporter (LUC) in yeast. Luciferase activity is given on a Log10 scale as relative counts of light emission per second (rcps) normalized to the cell density. The OST1-mediated induction factor is indicated above the columns. In the absence of ABFs, the LUC activity was 3 x 103 rcps.  Combining the ABA signaling components ABF2, OST1, the PP2C ABI1, and RCAR11 provides ABA-controlled LUC expression. The reporter is under the control of a minimal promoter that contains ABA-responsive cis elements (ABREs). Core ABA signaling involves the RCAR receptor that inhibits the ABA coreceptor (PP2C) in the presence of ABA and allows the activation of the protein kinase SnRK2. SnRK2 phosphorylates and activates the key transcription factor ABF, which targets promoters containing ABRE. Reporter activity was determined 16 h after exogenous ABA (0.1 mM) administration and referred to samples expressing ABF2 alone.


Figure 2 In yeast , the OST1 - and ABF2 - mediated reporter activity was inhibited by ABI1 ( ‑ RCAR ) . The subsequent recovery of the response was determined by co - expressed ABA receptors RCAR1 - RCAR14 belonging to three subfamilies , in the presence or absence of 30 µM ABA . The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A . Regulation of an ABA - responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1 - effector in combination with 5 µg ABA receptor DNA ( ± 10 µM ABA ) per sample containing 105 protoplasts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 In yeast, the OST1- and ABF2-mediated reporter activity was inhibited by ABI1 (‑RCAR). The subsequent recovery of the response was determined by co-expressed ABA receptors RCAR1-RCAR14 belonging to three subfamilies, in the presence or absence of 30 µM ABA. The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A. Regulation of an ABA-responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1-effector in combination with 5 µg ABA receptor DNA (± 10 µM ABA) per sample containing 105 protoplasts.


Figure 3 The ABA - dependent regulation of the PP2Cs ABI1 ( A , D , G ) , ABI2 ( B , E , H ) , and HAB1 ( C , F , I ) by RCAR12 and RCAR13 were analyzed in yeast ( A - C ) , Arabidopsis protoplasts ( D - F ) , and in vitro ( G - I ) . A - C ) The analysis in yeast was conducted as mentioned in Fig . 2A except that other PP2C - RCAR combinations were used and the ABA concentrations varied as indicated . D - F ) Analysis of protoplasts was performed in the ABA - deficient background aba2 - 1 in the presence of different exogenous ABA concentrations . The ABA response was approximately tenfold inhibited by ectopic expression of the respective PP2Cs ( Tischer et al , 2017 ) . The induction of the reporter gene is related to protoplasts without ectopic RCAR expression , and in the absence of exogenous ABA . G - I ) The in vitro analysis was performed by using 50 nM PP2C and 100 nM RCAR at different ABA concentrations . Maximum inhibition of PP2C activity by ABA was set to 100 % . Data information : In ( A - C ) each data point represents the mean ± s . d . ; n = 9 biological replicates derived from three independent yeast transformants . In ( D - F ) data represent the mean ± s . d . of three independent transfections . In ( G - I ) data represent the mean ± s . d . of three replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 The ABA-dependent regulation of the PP2Cs ABI1 (A,D,G), ABI2 (B,E,H), and HAB1 (C,F,I) by RCAR12 and RCAR13 were analyzed in yeast (A-C), Arabidopsis protoplasts (D-F), and in vitro (G-I). A-C) The analysis in yeast was conducted as mentioned in Fig. 2A except that other PP2C - RCAR combinations were used and the ABA concentrations varied as indicated. D-F) Analysis of protoplasts was performed in the ABA-deficient background aba2-1 in the presence of different exogenous ABA concentrations. The ABA response was approximately tenfold inhibited by ectopic expression of the respective PP2Cs (Tischer et al, 2017). The induction of the reporter gene is related to protoplasts without ectopic RCAR expression, and in the absence of exogenous ABA. G -I) The in vitro analysis was performed by using 50 nM PP2C and 100 nM RCAR at different ABA concentrations. Maximum inhibition of PP2C activity by ABA was set to 100%. Data information: In (A-C) each data point represents the mean ± s.d.; n = 9 biological replicates derived from three independent yeast transformants. In (D-F) data represent the mean ± s.d. of three independent transfections. In (G-I) data represent the mean ± s.d. of three replicates.


Figure 4 Expression of the SnRK2s in yeast revealed ABF2 - mediated transactivation by SnRK2 . 1 , SnRK2 . 4 , SnRK2 . 5 , and SnRK2 . 10 in addition to OST1 . Inset : Immuno - detection of FLAG - tagged SnRK2s expressed in yeast . The data in ( B ) are normalized to control samples expressing ABF2 but no SnRK2 The active subgroup I members together with SnRK2 . 2 and SnRK2 . 3 were analyzed for differential activation of ABFs . The OST1 data shown in Fig . 1A is included for better comparison . The induction level of ABF4 relative to the empty vector control ( ev ) is indicated . Analysis of the phosphorylation status in the activation loop of SnRK2s expressed in yeast . Phosphorylation occurred at serine 175 or threonine 176 , and at threonine 179 . The relative abundance is given as the ratio of signal intensity of phosphorylated versus non - phosphorylated peptide for OST1 , SnRK2 . 3 , and SnRK2 . 4 in the mass spectrometric analysis . The numbering of phosphosites corresponds to OST1 . Inhibition of OST1 and SnRK2 . 4 by clade A PP2Cs . The data in ( G ) to cells expressing inactive ABI1D177A instead of ABI1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 Expression of the SnRK2s in yeast revealed ABF2-mediated transactivation by SnRK2.1, SnRK2.4, SnRK2.5, and SnRK2.10 in addition to OST1. Inset: Immuno-detection of FLAG-tagged SnRK2s expressed in yeast. The data in (B) are normalized to control samples expressing ABF2 but no SnRK2  The active subgroup I members together with SnRK2.2 and SnRK2.3 were analyzed for differential activation of ABFs. The OST1 data shown in Fig. 1A is included for better comparison. The induction level of ABF4 relative to the empty vector control (ev) is indicated.  Analysis of the phosphorylation status in the activation loop of SnRK2s expressed in yeast. Phosphorylation occurred at serine 175 or threonine 176, and at threonine 179. The relative abundance is given as the ratio of signal intensity of phosphorylated versus non-phosphorylated peptide for OST1, SnRK2.3, and SnRK2.4 in the mass spectrometric analysis. The numbering of phosphosites corresponds to OST1.  Inhibition of OST1 and SnRK2.4 by clade A PP2Cs. The data in (G) to cells expressing inactive ABI1D177A instead of ABI1.


Figure 5A , B ) ABF2 - mediated reporter induction by different SnRK2s was assessed in the presence of the receptor complex RCAR11 and ABI1 , and varying exogenous ABA concentrations . Data information : The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines . represent Replicates were normalized to controls without SnRK2 in ( A , B ) C ) Maximum ABA response recovery of samples shown in ( A , B ) relative to the reporter activity of 0 . 1 mM ABA - treated yeast cells expressing SnRK2s but no ABI1 . Data information : Replicates were normalized to controls without ABI1 and RCAR for each SnRK2 in ( C ) . D - F ) Analysis of the ABA response mediated by OST1 and SnRK2 . 4 using the receptors RCAR1 ( D ) , RCAR8 ( E ) , and RCAR14 ( F ) . Data information : The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines . The data in ( D - F ) are the mean ± s . e . m ; n = 12 , biological replicates from four independent yeast transformants for OST1 + RCAR1 / RCAR8 , and SnRK2 . 4 + RCAR8 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A,B) ABF2-mediated reporter induction by different SnRK2s was assessed in the presence of the receptor complex RCAR11 and ABI1, and varying exogenous ABA concentrations. Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. represent Replicates were normalized to controls without SnRK2 in (A, B) C) Maximum ABA response recovery of samples shown in (A,B) relative to the reporter activity of 0.1 mM ABA-treated yeast cells expressing SnRK2s but no ABI1. Data information: Replicates were normalized to controls without ABI1 and RCAR for each SnRK2 in (C).  D-F) Analysis of the ABA response mediated by OST1 and SnRK2.4 using the receptors RCAR1 (D), RCAR8 (E), and RCAR14 (F). Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. The data in (D-F) are the mean ± s.e.m; n = 12, biological replicates from four independent yeast transformants for OST1 + RCAR1/RCAR8, and SnRK2.4 + RCAR8.


Figure 6FRET - FLIM analysis of GFP : OST1 or GFP : SnRK2 . 4 and the effect of ABI1 fused to the mCherry fluorophore in the presence of RCAR1 and ABA . GFP lifetimes were determined after transient expression of the Arabidopsis proteins in leaves of Nicotiana benthamiana . Representative false colour images of GFP : SnRK2 lifetimes measured in leaf tissues expressing OST1 or SnRK2 . 4 in combination with mCherry : ABI1 and RCAR1 without exogenous ABA . The bar marks 20 µm and the nucleus ( n ) and cytosol ( c ) are indicated . Analysis of SnRK2 - ABI1 interaction as mentioned in ( A ) but in the presence of RCAR14 and different exogenous ABA levels . RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1 . Representative GFP lifetime false colour image of the analysis shown in C ) 2h after 30 µM exogenous ABA administration . Details are as described in ( B ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6FRET-FLIM analysis of GFP:OST1 or GFP:SnRK2.4 and the effect of ABI1 fused to the mCherry fluorophore in the presence of RCAR1 and ABA. GFP lifetimes were determined after transient expression of the Arabidopsis proteins in leaves of Nicotiana benthamiana.   Representative false colour images of GFP:SnRK2 lifetimes measured in leaf tissues expressing OST1 or SnRK2.4 in combination with mCherry:ABI1 and RCAR1 without exogenous ABA. The bar marks 20 µm and the nucleus (n) and cytosol (c) are indicated.    Analysis of SnRK2-ABI1 interaction as mentioned in (A) but in the presence of RCAR14 and different exogenous ABA levels. RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1.   Representative GFP lifetime false colour image of the analysis shown in C) 2h after 30 µM exogenous ABA administration. Details are as described in (B).


Figure 1A ) ( left ) t - sne plot from scRNAseq data of human organoids and cluster assignment of colonic cell populations . Red color corresponds to undifferentiated cells , light green corresponds to surface colonocytes ( enterocytes ) . t - sne plot for selected genes from single cell RNAseq of human colon epithelial cells revealing expression of ACE2 specifically in differentiated surface colonocytes expressing KRT20 and AQP8 , which also express the IFN - γ receptor IFNGR2 . B ) Immunofluorescence staining for ACE2 ( red ) and KRT20 ( green ) of human colon tissue section indicating ACE2 expression in surface colonocytes . Scale bar : 50 µm . C ) Human colon organoids cultured in full medium ( FM ) condition ( upper panel ) or FM treated with IFN - γ ( lower panel ) . Left : bright field images of 7 - day cultured human colon organoids , scale bar : 100 µm . Right : Immunofluorescence for KRT20 ( green ; note increased expression upon IFN - γ treatment ) . Scale bar : 25 µm . D ) Human colon organoids cultured in basic medium ( upper panel ) or additionally treated with IFN - γ ( lower panel ) . Left : bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN - γ ( lower panel ) scale bar : 100 µm . Middle : Immunofluorescence labelling for KRT20 ( green ) Right : Immunofluorescence labelling for E - cadherin ( green ) to visualize epithelial cell boundaries ; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN - γ . Scale bar : 25 µm . Comparison of E ) KRT20 , F ) MUC2 , G ) CHGA , H ) LGR5 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons testACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons testJ ) Immunofluorescence staining for ACE2 ( red ) and KRT20 ( green ) of organoids grown in full medium ( upper ) or basic medium ( lower ) and additionally treated with IFN - γ ( right ) , indicating expression of ACE2 in differentiated enterocytes . White arrows point to ACE2 . Scale bar : 10

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) (left) t-sne plot from scRNAseq data of human organoids and cluster assignment of colonic cell populations. Red color corresponds to undifferentiated cells, light green corresponds to surface colonocytes (enterocytes). t-sne plot for selected genes from single cell RNAseq of human colon epithelial cells revealing expression of ACE2 specifically in differentiated surface colonocytes expressing KRT20 and AQP8, which also express the IFN-γ receptor IFNGR2. B) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of human colon tissue section indicating ACE2 expression in surface colonocytes. Scale bar: 50 µm.C) Human colon organoids cultured in full medium (FM) condition (upper panel) or FM treated with IFN-γ (lower panel). Left: bright field images of 7-day cultured human colon organoids, scale bar: 100 µm. Right: Immunofluorescence for KRT20 (green; note increased expression upon IFN-γ treatment). Scale bar: 25 µm.D) Human colon organoids cultured in basic medium (upper panel) or additionally treated with IFN-γ (lower panel). Left: bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN-γ (lower panel) scale bar: 100 µm. Middle: Immunofluorescence labelling for KRT20 (green) Right: Immunofluorescence labelling for E-cadherin (green) to visualize epithelial cell boundaries; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN-γ. Scale bar: 25 µm.Comparison of E) KRT20, F) MUC2, G) CHGA, H) LGR5 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons testACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons testJ) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of organoids grown in full medium (upper) or basic medium (lower) and additionally treated with IFN-γ (right), indicating expression of ACE2 in differentiated enterocytes. White arrows point to ACE2. Scale bar: 10 µm


Figure 2A ) Immunofluorescence for N - protein ( green ) 48 h after SARS - CoV - 2 infection of human colon organoids cultured in full medium ( upper ) , full medium + IFN - γ ( upper right ) , basic medium ( lower left ) and basic medium + IFN - γ ( lower right ) . Yellow arrows point to N - protein positive cells . Scale bar : 25 µm . B ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium vs . basic medium organoids 48 h after infection and normalized to GAPDH ( n = 6 ) . C ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium vs . full medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 9 ) . D ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in basic medium vs . basic medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 4 ) . E ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium + IFN - γ vs . basic medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 4 ) . ( B - E ) Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by Student ' s t - test F ) qPCR data comparing the relative virus load of SARS - CoV - 2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium ( left ) and basic medium ( right ) untreated or treated with IFN - γ , indicating increase in virus load in differentiated and IFN - γ treated conditions ( n = 8 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons testG ) Electron microscopy imaging of organoids grown in basic medium , pretreated with IFN - γ for 3 days and infected for 48 h with SARS - CoV - 2 . Upper : Overview showing large virus - loaded vesicles in an epithelial cell . Scale bar : 500nm . Lower : Disintegrated organoid cell containing high virus load , scale bar : 2 . 5µm , higher magnification to visualize virus particles , scale bar : 100nm . H ) Immunofluorescence for cleaved - caspase3 ( red ) and E - cadherin ( green ) 48 h after SARS - CoV - 2 infection of human colon organoids cultured in full medium ( upper ) , full medium + IFN - γ ( upper right ) , basic medium ( lower left ) and basic medium + IFN - γ ( lower right ) . Scale bar : 50 µm . ( below ) Quantification of apoptotic cells , indicating increased cell death in the differentiated and IFN - γ treated condition after SARS - CoV - 2 infection ( n = 7 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) Immunofluorescence for N-protein (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Yellow arrows point to N-protein positive cells. Scale bar: 25 µm.B) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. basic medium organoids 48 h after infection and normalized to GAPDH (n=6). C) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. full medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=9). D) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in basic medium vs. basic medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). E) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium + IFN-γ vs. basic medium+ IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). (B-E) Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by Student's t-test F) qPCR data comparing the relative virus load of SARS-CoV-2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium (left) and basic medium (right) untreated or treated with IFN-γ, indicating increase in virus load in differentiated and IFN-γ treated conditions (n=8). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons testG) Electron microscopy imaging of organoids grown in basic medium, pretreated with IFN-γ for 3 days and infected for 48 h with SARS-CoV-2. Upper: Overview showing large virus-loaded vesicles in an epithelial cell. Scale bar: 500nm. Lower: Disintegrated organoid cell containing high virus load, scale bar: 2.5µm, higher magnification to visualize virus particles, scale bar: 100nm.H) Immunofluorescence for cleaved-caspase3 (red) and E-cadherin (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Scale bar: 50 µm. (below) Quantification of apoptotic cells, indicating increased cell death in the differentiated and IFN-γ treated condition after SARS-CoV-2 infection (n=7). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


Figure 3C ) Analysis of discordance and concordance of transcriptomic responses ( DISCO ) between untreated vs . SARS - CoV - 2 infected and untreated vs IFN - γ treated organoids . The 50 most regulated genes are highlighted ; grey : discordant and black : concordant . D ) Relative expression of selected enterocyte - specific genes upon SARS - CoV - 2 infection , normalized to GAPDH . Differences in gene expression were assessed using the linear model ' lmFit ' and ' makeContrasts ' in limma . E ) Immunofluorescence for KRT20 ( green ) in human colon organoids cultured in basic medium and either non - infected ( left ) or 48 h after SARS - CoV - 2 infection ( right ) , indicating a strong increase in KRT20 expression after viral infection . Scale bar : 50 µm . Quantification of fluorescence intensity below ( n = 8 ) . Data are presented as mean + / - SD , * * * * : p ≤ 0 . 0001 , as determined by Student ' s t - testF ) qPCR data displaying the increase of INF - γ target genes ( CXCL11 , IFIT3 ) ( upper ) and enterocyte marker genes ( KRT20 , ACE2 ) ( lower ) after SARS - CoV - 2 infection , validating the results from the microarray analysis ( n = 3 ) . Data are presented as mean + / - SD , * * * * : p ≤ 0 . 0001 , as determined by Student ' s t -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C) Analysis of discordance and concordance of transcriptomic responses (DISCO) between untreated vs. SARS-CoV-2 infected and untreated vs IFN-γ treated organoids. The 50 most regulated genes are highlighted; grey: discordant and black: concordant.D) Relative expression of selected enterocyte-specific genes upon SARS-CoV-2 infection, normalized to GAPDH. Differences in gene expression were assessed using the linear model 'lmFit' and 'makeContrasts' in limma.E) Immunofluorescence for KRT20 (green) in human colon organoids cultured in basic medium and either non-infected (left) or 48 h after SARS-CoV-2 infection (right), indicating a strong increase in KRT20 expression after viral infection. Scale bar: 50 µm. Quantification of fluorescence intensity below (n=8). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-testF) qPCR data displaying the increase of INF-γ target genes (CXCL11, IFIT3) (upper) and enterocyte marker genes (KRT20, ACE2) (lower) after SARS-CoV-2 infection, validating the results from the microarray analysis (n=3). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test


Figure 1Montages of representative DAPI - stained nuclei are shown for gene knockdowns that led to reduced nuclear size . Cell - to - cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability . Ten nuclei per column , organized by maximum nuclear size z - score . Scale bar , 25 µm . Median nuclear size z - scores are plotted for gene knockdowns that led to reduced nuclear size . Data are based on three different siRNA oligo sequences for each gene and two biological replicates . Error bars represent the SEM for biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Montages of representative DAPI-stained nuclei are shown for gene knockdowns that led to reduced nuclear size. Cell-to-cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability. Ten nuclei per column, organized by maximum nuclear size z-score. Scale bar, 25 µm. Median nuclear size z-scores are plotted for gene knockdowns that led to reduced nuclear size. Data are based on three different siRNA oligo sequences for each gene and two biological replicates. Error bars represent the SEM for biological replicates.


Figure 2MCF - 10AT1k . cl2 cells were transfected with control or ELYS siRNA . ( A ) Representative images of cells stained for lamin B2 and B1 are shown . ( B ) Representative images of cells stained for lamin A / C and B1 are shown . ( C ) For each experiment , nuclear lamin B1 , B2 , and A / C staining intensities were quantified for 44 - 159 nuclei per condition ( 96 nuclei on average ) and normalized to the negative control . Three biological replicates , data from one representative experiment shown . ( D ) Representative images of cells stained for FG - Nups ( mAb414 ) and lamin B1 are shown . ( E ) Representative images of cells stained for Nup133 and lamin B1 are shown . ( F ) For each experiment , nuclear mAb414 and Nup133 staining intensities were quantified for 31 - 83 nuclei per condition ( 60 nuclei on average ) and normalized to the negative control . To measure NPC densities , confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei . NPC numbers were counted per unit area for 18 - 50 nuclei per condition ( 30 nuclei on average ) and normalized to the negative control . Two biological replicates , data from one representative experiment shown . MCF - 10AT1k . cl2 cells were transfected with control or ELYS siRNA . Representative images of cells stained for Ran and lamin B1 are shown . ( H ) Representative images of cells stained for NTF2 and lamin A / C are shown . ( I ) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the negative control . To measure the N / C staining intensity ratio of a given cell , the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region . For each experiment , 54 - 216 nuclei were quantified per condition ( 112 nuclei on average ) . Two biological replicates , data from one representative experiment shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2MCF-10AT1k.cl2 cells were transfected with control or ELYS siRNA. (A) Representative images of cells stained for lamin B2 and B1 are shown. (B) Representative images of cells stained for lamin A/C and B1 are shown. (C) For each experiment, nuclear lamin B1, B2, and A/C staining intensities were quantified for 44-159 nuclei per condition (96 nuclei on average) and normalized to the negative control. Three biological replicates, data from one representative experiment shown. (D) Representative images of cells stained for FG-Nups (mAb414) and lamin B1 are shown. (E) Representative images of cells stained for Nup133 and lamin B1 are shown. (F) For each experiment, nuclear mAb414 and Nup133 staining intensities were quantified for 31-83 nuclei per condition (60 nuclei on average) and normalized to the negative control. To measure NPC densities, confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei. NPC numbers were counted per unit area for 18-50 nuclei per condition (30 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.MCF-10AT1k.cl2 cells were transfected with control or ELYS siRNA. Representative images of cells stained for Ran and lamin B1 are shown. (H) Representative images of cells stained for NTF2 and lamin A/C are shown. (I) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the negative control. To measure the N/C staining intensity ratio of a given cell, the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region. For each experiment, 54-216 nuclei were quantified per condition (112 nuclei on average). Two biological replicates, data from one representative experiment shown.


Figure 3MCF - 10AT1k . cl2 cells were co - transfected with control or ELYS siRNA and plasmids expressing eGFP alone or siRNA - resistant eGFP - ELYS . Cell lysates were analyzed by western blot and probed for ELYS and tubulin . ELYS band intensity was normalized to tubulin and then normalized to the eGFP / negative siRNA control . ELYS concentrations are indicated in the row labeled [ ELYS ] ( average ± SD ) . Quantification was performed on two biological replicates . Cells were stained with an antibody against lamin B1 . Representative images are shown . ( B ) For each experiment , nuclear cross - sectional areas were quantified for 68 - 298 nuclei per condition ( 162 nuclei on average ) and averaged . Three biological replicates , data from one representative experiment shown . ( C ) For each experiment , nuclear lamin B2 staining intensities were quantified for 29 - 81 nuclei per condition ( 60 nuclei on average ) and normalized to the eGFP / negative siRNA control . Two biological replicates , data from one representative experiment shown . Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated . For each experiment , 58 - 299 cells were examined per condition . Three biological replicates , data from one representative experiment shown . Cells were stained with antibodies against Ran and NTF2 . For each experiment , 62 - 130 nuclei were quantified per condition ( 92 nuclei on average ) . Two biological replicates , data from one representative experiment shown . ( E ) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the eGFP control . ( F ) To measure the N / C staining intensity ratio of a given cell , the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region . Transfections were performed as in ( A ) and cells were stained with an antibody against FG - Nups ( mAb414 ) and Nup133 . Representative images for mAb414 staining are shown . Confocal imaging was performed through nuclear cross - sections as well as on the surface of nuclei . ( H ) To measure NPC densities , confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei . NPC numbers were counted per unit area for 25 - 116 nuclei per condition ( 53 nuclei on average ) and normalized to the negative control . Two biological replicates , data from one representative experiment shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3MCF-10AT1k.cl2 cells were co-transfected with control or ELYS siRNA and plasmids expressing eGFP alone or siRNA-resistant eGFP-ELYS. Cell lysates were analyzed by western blot and probed for ELYS and tubulin. ELYS band intensity was normalized to tubulin and then normalized to the eGFP/negative siRNA control. ELYS concentrations are indicated in the row labeled [ELYS] (average ± SD). Quantification was performed on two biological replicates. Cells were stained with an antibody against lamin B1. Representative images are shown. (B) For each experiment, nuclear cross-sectional areas were quantified for 68-298 nuclei per condition (162 nuclei on average) and averaged. Three biological replicates, data from one representative experiment shown. (C) For each experiment, nuclear lamin B2 staining intensities were quantified for 29-81 nuclei per condition (60 nuclei on average) and normalized to the eGFP/negative siRNA control. Two biological replicates, data from one representative experiment shown.Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 58-299 cells were examined per condition. Three biological replicates, data from one representative experiment shown.Cells were stained with antibodies against Ran and NTF2. For each experiment, 62-130 nuclei were quantified per condition (92 nuclei on average). Two biological replicates, data from one representative experiment shown. (E) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the eGFP control. (F) To measure the N/C staining intensity ratio of a given cell, the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region.Transfections were performed as in (A) and cells were stained with an antibody against FG-Nups (mAb414) and Nup133. Representative images for mAb414 staining are shown. Confocal imaging was performed through nuclear cross-sections as well as on the surface of nuclei. (H) To measure NPC densities, confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei. NPC numbers were counted per unit area for 25-116 nuclei per condition (53 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.


Figure 4MCF - 10AT1k . cl2 cells were co - transfected with control or ELYS siRNA and plasmids expressing mCherry alone or mCherry - importin α . Cells were stained with an antibody against lamin B2 . Representative images are shown . Scale bar , 10 µm . ( B ) For each experiment , nuclear cross - sectional areas were quantified for 30 - 172 nuclei per condition ( 84 nuclei on average ) and averaged . Two biological replicates , data from one representative experiment shown . Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated . For each experiment , 74 - 218 cells were examined per condition . Two biological replicates , data from one representative experiment shown . For each experiment , nuclear lamin B2 staining intensities were quantified for 34 - 187 nuclei per condition ( 85 nuclei on average ) and normalized to the mCherry / negative siRNA control . To measure bulk import capacity for importin α / β cargos , cells were co - transfected as in ( A ) along with a plasmid expressing GFP - 3x SV40 NLS . For each experiment , nuclear GFP - NLS intensities were quantified for 22 - 188 nuclei per condition ( 73 nuclei on average ) and normalized to the mCherry / negative siRNA control . Two biological replicates , data from one representative experiment shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4MCF-10AT1k.cl2 cells were co-transfected with control or ELYS siRNA and plasmids expressing mCherry alone or mCherry-importin α. Cells were stained with an antibody against lamin B2. Representative images are shown. Scale bar, 10 µm. (B) For each experiment, nuclear cross-sectional areas were quantified for 30-172 nuclei per condition (84 nuclei on average) and averaged. Two biological replicates, data from one representative experiment shown.Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 74-218 cells were examined per condition. Two biological replicates, data from one representative experiment shown.For each experiment, nuclear lamin B2 staining intensities were quantified for 34-187 nuclei per condition (85 nuclei on average) and normalized to the mCherry/negative siRNA control. To measure bulk import capacity for importin α/β cargos, cells were co-transfected as in (A) along with a plasmid expressing GFP-3x SV40 NLS. For each experiment, nuclear GFP-NLS intensities were quantified for 22-188 nuclei per condition (73 nuclei on average) and normalized to the mCherry/negative siRNA control. Two biological replicates, data from one representative experiment shown.


Figure 5MCF - 10AT1k . cl2 cells were transfected with plasmids expressing mCherry alone or mCherry - RanQ69L and stained with an antibody against lamin B2 . Representative images are shown . MCF - 10AT1k . cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1 . Representative images are shown . For each experiment , nuclear cross - sectional areas were quantified for 32 - 271 nuclei per condition ( 123 nuclei on average ) and averaged . Three biological replicates , data from one representative experiment shown . To measure bulk import capacity for importin α / β cargos , experiments were performed as in ( A - B ) except that cells were also transfected with a plasmid expressing GFP - 3x SV40 NLS . For each experiment , nuclear GFP - NLS intensities were quantified for 11 - 75 nuclei per condition ( 35 nuclei on average ) and normalized to the appropriate control . Two biological replicates , data from one representative experiment shown . Cells with and without cytoplasmic lamin B aggregates were counted and the percentage of cells with aggregates was calculated . For each experiment , 31 - 130 cells were examined per condition . Three biological replicates , data from one representative experiment shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5MCF-10AT1k.cl2 cells were transfected with plasmids expressing mCherry alone or mCherry-RanQ69L and stained with an antibody against lamin B2. Representative images are shown.MCF-10AT1k.cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1. Representative images are shown.For each experiment, nuclear cross-sectional areas were quantified for 32-271 nuclei per condition (123 nuclei on average) and averaged. Three biological replicates, data from one representative experiment shown.To measure bulk import capacity for importin α/β cargos, experiments were performed as in (A-B) except that cells were also transfected with a plasmid expressing GFP-3x SV40 NLS. For each experiment, nuclear GFP-NLS intensities were quantified for 11-75 nuclei per condition (35 nuclei on average) and normalized to the appropriate control. Two biological replicates, data from one representative experiment shown.Cells with and without cytoplasmic lamin B aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 31-130 cells were examined per condition. Three biological replicates, data from one representative experiment shown.


Figure 6Cell lysates from control and XPO1 siRNA transfected MCF - 10AT1k . cl2 cells were analyzed by western blot and probed for XPO1 and tubulin . One representative western blot is shown . XPO1 band intensity was normalized to tubulin . Quantification from two biological replicates is shown . MCF - 10AT1k . cl2 cells were transfected with control or XPO1 siRNA . Lamin B1 and lamin A / C nuclear staining intensities were quantified based on experiments using two different XPO1 siRNA sequences and two biological replicates . For each experiment , 108 - 865 nuclei were quantified per condition ( 416 nuclei on average ) . Average and SD values based on averages from these four experiments are plotted , normalized to the negative siRNA control . MCF - 10AT1k . cl2 cells were transfected with the indicated siRNAs and nuclear cross - sectional areas were quantified . For each experiment , 203 - 604 nuclei were quantified per condition ( 403 nuclei on average ) . Average and SD values based on averages from four biological replicates are plotted , normalized to the negative siRNA control . MCF - 10AT1k . cl2 cells were transfected with control siRNA or with siRNA against XPO1 . Cells were stained with DAPI and quantification of DNA staining intensity was used to estimate the fraction of cells in various stages of the cell cycle by high - throughput imaging as previously described [ 123 ] ( see Methods ) . The stacked bars represent the means of the fractions for each cell cycle phase calculated over 3 biological replicates . MCF - 10AT1k . cl2 cells were transfected with control or XPO1 siRNA . For the bottom row of images , cells transfected with control siRNA were treated with 20 ng / ml leptomycin B for 24 hours . Cells were fixed and stained with 2 µg / ml FITC . Representative images are shown . Scale bar , 20 µm . For each experiment , nuclear cross - sectional areas and nuclear FITC staining intensities were quantified for 105 - 620 nuclei per condition ( 267 nuclei on average ) and normalized to the negative control . Two biological replicates , data from one representative experiment shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Cell lysates from control and XPO1 siRNA transfected MCF-10AT1k.cl2 cells were analyzed by western blot and probed for XPO1 and tubulin. One representative western blot is shown. XPO1 band intensity was normalized to tubulin. Quantification from two biological replicates is shown.MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. Lamin B1 and lamin A/C nuclear staining intensities were quantified based on experiments using two different XPO1 siRNA sequences and two biological replicates. For each experiment, 108-865 nuclei were quantified per condition (416 nuclei on average). Average and SD values based on averages from these four experiments are plotted, normalized to the negative siRNA control.MCF-10AT1k.cl2 cells were transfected with the indicated siRNAs and nuclear cross-sectional areas were quantified. For each experiment, 203-604 nuclei were quantified per condition (403 nuclei on average). Average and SD values based on averages from four biological replicates are plotted, normalized to the negative siRNA control.MCF-10AT1k.cl2 cells were transfected with control siRNA or with siRNA against XPO1. Cells were stained with DAPI and quantification of DNA staining intensity was used to estimate the fraction of cells in various stages of the cell cycle by high-throughput imaging as previously described [123] (see Methods). The stacked bars represent the means of the fractions for each cell cycle phase calculated over 3 biological replicates.MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. For the bottom row of images, cells transfected with control siRNA were treated with 20 ng/ml leptomycin B for 24 hours. Cells were fixed and stained with 2 µg/ml FITC. Representative images are shown. Scale bar, 20 µm. For each experiment, nuclear cross-sectional areas and nuclear FITC staining intensities were quantified for 105-620 nuclei per condition (267 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.


Figure 1 ( B ) COUP - TFII expression ( green ) is increased in human kidneys from patients with thrombotic microangiopathy ( TMA ) and diabetic nephropathy ( DN ) ( n = 2 ) . Quantification by confocal micrographs in 400x hpf . * * * * p < 0 . 0001 by one - way ANOVA ; mean ± SD . The majority of these cells were localized in the interstitial region . More than 50 % COUP - TFII ( + ) cells ( green ) are co - localized with expanded αSMA - positive ( red ) areas of fibrosis . Pearson correlation coefficient ( PCC ) = 0 . 56 ± 0 . 1 for patient # 1 and 0 . 65 ± 0 . 13 for patient # 2 . ( C ) COUP - TFII expression ( green ) is increased in lungs from patients with IPF ( n = 2 ) by immunofluorescence . Quantification by confocal micrographs in 400x hpf . * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD . ( E ) COUP - TFII expression increased significantly in organoids treated with TGFβ1 ( 10ng / ml ) , cobalt chloride ( 100 μM ) , IL - 1β ( 10ng / ml ) , but not PDGF ( 50ng / ml ) . Quantification by confocal micrographs in 400x hpf . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD . The higher magnification of the insert from IL - 1β φησwed co - localization of COUP - TFII ( green ) and αSMA ( red ) staining .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) COUP-TFII expression (green) is increased in human kidneys from patients with thrombotic microangiopathy (TMA) and diabetic nephropathy (DN) (n=2). Quantification by confocal micrographs in 400x hpf. ****p<0.0001 by one-way ANOVA; mean ± SD. The majority of these cells were localized in the interstitial region. More than 50% COUP-TFII (+) cells (green) are co-localized with expanded αSMA-positive (red) areas of fibrosis. Pearson correlation coefficient (PCC) = 0.56 ± 0.1 for patient#1 and 0.65 ± 0.13 for patient#2.(C) COUP-TFII expression (green) is increased in lungs from patients with IPF (n=2) by immunofluorescence. Quantification by confocal micrographs in 400x hpf. **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD.(E) COUP-TFII expression increased significantly in organoids treated with TGFβ1 (10ng/ml), cobalt chloride (100 μM), IL-1β (10ng/ml), but not PDGF (50ng/ml). Quantification by confocal micrographs in 400x hpf. *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. The higher magnification of the insert from IL-1β φησwed co-localization of COUP-TFII (green) and αSMA (red) staining.