Figure 2 ( D ) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 ( shArfGAP1 ) or GFP ( shGFP ) control . Cells were cultured under normal condition ( NC ; lanes 1 and 7 ) , or starved of amino acids for 1 hour ( - AA 1h ; lanes 2 and 8 ) , followed by either maintaining in amino acid starvation media ( - AA ; lanes 3 and 9 ) , or addition of 1 mM of Leu ( + Leu ; lanes 4 and 10 ) , Gln ( + Gln ; lanes 5 and 11 ) , or Asn ( + Asn ; lanes 6 and 12 ) for 2 hours . mTORC1 activity was analyzed similar to ( B ) . Protein levels of ArfGAP1 were confirmed by immunoblotting . S6K1 and Actin were probed as controls . ( E ) RagA / B KO HEK293A cells were co - transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) with Myc - tagged S6K1 . Cells were starved of amino acids ( - AA ) for 2 hours and then 4mM of Gln or Asn were added for 2 hours . mTORC1 was analyzed similar to ( B ) . IP or WCL samples were probed for HA , Myc , and phosphorylated S6K1 at Thr 389 . Actin serve as a loading control .

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 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

Figure 2(D) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 (shArfGAP1) or GFP (shGFP) control. Cells were cultured under normal condition (NC; lanes 1 and 7), or starved of amino acids for 1 hour (-AA 1h; lanes 2 and 8), followed by either maintaining in amino acid starvation media (-AA; lanes 3 and 9), or addition of 1 mM of Leu (+Leu; lanes 4 and 10), Gln (+Gln; lanes 5 and 11), or Asn (+Asn; lanes 6 and 12) for 2 hours. mTORC1 activity was analyzed similar to (B). Protein levels of ArfGAP1 were confirmed by immunoblotting. S6K1 and Actin were probed as controls.(E) RagA/B KO HEK293A cells were co-transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4mM of Gln or Asn were added for 2 hours. mTORC1 was analyzed similar to (B). IP or WCL samples were probed for HA, Myc, and phosphorylated S6K1 at Thr 389. Actin serve as a loading control.


Figure 3 ( B ) Fluorescence Lifetime Imaging Microscopy ( FLIM ) and Förster resonance energy transfer ( FRET ) analysis were performed in wildtype ( WT , left ) or RagA / B KO ( middle ) HAP1 cells expressing mCherry - tagged ArfGAP1 and endogenously GFP - tagged Raptor . Cells either had amino acids ( + AA ) or were starved of amino acids ( - AA ) . At least 20 cells were analyzed in each group . Box plots shows the percentage of pixels with ≥ 30 % FRET efficiency of co - transfected cells . RFP1 was expressed and analyzed as a negative control . The whiskers of the boxplot represent the minimum and maximum values , while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median . Right - Expression level of RagA / B was confirmed Western blot analysis . Actin was used as a control .

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Figure 3(B) Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster resonance energy transfer (FRET) analysis were performed in wildtype (WT, left) or RagA/B KO (middle) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). At least 20 cells were analyzed in each group. Box plots shows the percentage of pixels with ≥ 30% FRET efficiency of co-transfected cells. RFP1 was expressed and analyzed as a negative control. The whiskers of the boxplot represent the minimum and maximum values, while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median. Right- Expression level of RagA/B was confirmed Western blot analysis. Actin was used as a control.


Figure 4 ( A ) Mouse embryonic fibroblast ( MEF ) cells were transfected with HA - tagged ArfGAP1 . Immunofluorescence staining was assessed for HA - tagged ArfGAP1 ( blue ) , mTOR ( green ) , and LAMP2 ( red , lysosomal marker ) . Left - A representative example of the immunofluorescence staining ( scale bar , 10 μm ) . The top left image shows the staining of HA - tagged ArfGAP1 , with a white outline highlighting the region that is positive for HA - tagged ArfGAP1 . This outline was overlaid with the mTOR channel ( top right ) and the LAMP2 channel ( bottom left ) . The mTOR and LAMP2 channels were then merged and processed using the Squassh segmentation algorithm ( Rizk et al . , 2014 ; bottom right ) . The co - localization of mTOR and LAMP2 was analyzed for the region within the outline ( HA - ArfGAP1 positive ) and the region outside of the outline ( HA - ArfGAP1 - negative ) , respectively . Right - In each microscopy field , the image was processed as described above , and the co - localization between mTOR and LAMP2 was quantified and normalized by LAMP2 positive puncta using the Squassh algorithm ( Rizk et al . , 2014 ) . The quantification was performed for the subgroups of cells that are positively or negatively stained for HA - tagged ArfGAP1 , respectively , and compared using Student ' s t - test . Columns represent Mean ± SEM ( N = 3 ) . ( D ) MIA Paca - 2 cells stably expressing FLAG - tagged or HA - tagged TMEM192 cells were cultured under normal condition ( NC ) or starved of amino acids ( - AA ) , and then stimulated with 2 mM glutamine and asparagine ( + Gln / Asn ) for 2 hours . HA - tagged TMEM192 was immunoprecipitated ( IP ) with HA beads and IP and whole cell lysates ( WCL ) were probed for ArfGAP1 , HA , RagA , or p18 ( LAMTOR1 ) . Flag , pS6K1 ( mTORC1 activity ) , S6K1 , and Vinculin were probed for as controls .

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 O O O O O 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 4(A) Mouse embryonic fibroblast (MEF) cells were transfected with HA-tagged ArfGAP1. Immunofluorescence staining was assessed for HA-tagged ArfGAP1 (blue), mTOR (green), and LAMP2 (red, lysosomal marker). Left- A representative example of the immunofluorescence staining (scale bar, 10 μm). The top left image shows the staining of HA-tagged ArfGAP1, with a white outline highlighting the region that is positive for HA-tagged ArfGAP1. This outline was overlaid with the mTOR channel (top right) and the LAMP2 channel (bottom left). The mTOR and LAMP2 channels were then merged and processed using the Squassh segmentation algorithm (Rizk et al., 2014; bottom right). The co-localization of mTOR and LAMP2 was analyzed for the region within the outline (HA-ArfGAP1 positive) and the region outside of the outline (HA-ArfGAP1-negative), respectively. Right- In each microscopy field, the image was processed as described above, and the co-localization between mTOR and LAMP2 was quantified and normalized by LAMP2 positive puncta using the Squassh algorithm (Rizk et al., 2014). The quantification was performed for the subgroups of cells that are positively or negatively stained for HA-tagged ArfGAP1, respectively, and compared using Student's t-test. Columns represent Mean ± SEM (N = 3).(D) MIA Paca-2 cells stably expressing FLAG-tagged or HA-tagged TMEM192 cells were cultured under normal condition (NC) or starved of amino acids (-AA), and then stimulated with 2 mM glutamine and asparagine (+Gln/Asn) for 2 hours. HA-tagged TMEM192 was immunoprecipitated (IP) with HA beads and IP and whole cell lysates (WCL) were probed for ArfGAP1, HA, RagA, or p18 (LAMTOR1). Flag, pS6K1 (mTORC1 activity), S6K1, and Vinculin were probed for as controls.


Figure 5 ( D ) Top - Input of GST , GST - tagged ArfGAP1 , and GST - tagged ArfGAP1 L207D / V279D that was us in the in vitro binding assays . Bottom - Flag - tagged mTOR , Flag - tagged Raptor , and Flag - tagged mLST8 were overexpressed in HEK293A cells . Cell lysates were immunoprecipitated ( IP ) with anti - Flag beads and incubated with either GST ( control ) , GST - tagged ArfGAP1 , or GST - ArfGAP1 L207D / V279D . IP or WCL samples were probed for Flag or GST .

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Figure 5(D) Top - Input of GST, GST-tagged ArfGAP1, and GST-tagged ArfGAP1 L207D/V279D that was us in the in vitro binding assays. Bottom - Flag-tagged mTOR, Flag-tagged Raptor, and Flag-tagged mLST8 were overexpressed in HEK293A cells. Cell lysates were immunoprecipitated (IP) with anti-Flag beads and incubated with either GST (control), GST-tagged ArfGAP1, or GST-ArfGAP1 L207D/V279D. IP or WCL samples were probed for Flag or GST.


Figure 6 ( A ) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of ARFGAP1 mRNA expression . Overall survival and disease - free survival were compared between these two groups , as shown in Kaplan - Meier curves . Median survivals ( in months ) , log - rank P values , and hazard ratios ( HR ) with 95 % confidence intervals are indicated . ( D ) MIA Paca - 2 cells stably expressing short hairpin RNAs ( shRNAs ) against ArfGAP1 or GFP ( control ) were plated and treated with mTOR inhibitor Torin 1 ( 250 nM ) or vehicle control for 48 hours prior to cell counting . Columns represent Mean ± SEM ( N = 6 ) . Student ' s t - test shGFP Vehicle vs . shGFP Torin 1 ( P = 2 . 83 E - 07 ) , shGFP Vehicle vs . shArfGAP1 Vehicle ( * P = 0 . 025 ) , shGFP Torin 1 vs , shArfGAP1 Torin 1 ( P = 0 . 139 ) , and shArfGAP1 Vehicle vs . shArfGAP1 Torin 1 ( P = 1 . 22 E - 07 ) . " ns " stands for " not significant " .

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 O O O 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 6(A) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of ARFGAP1 mRNA expression. Overall survival and disease-free survival were compared between these two groups, as shown in Kaplan-Meier curves. Median survivals (in months), log-rank P values, and hazard ratios (HR) with 95% confidence intervals are indicated.(D) MIA Paca-2 cells stably expressing short hairpin RNAs (shRNAs) against ArfGAP1 or GFP (control) were plated and treated with mTOR inhibitor Torin 1 (250 nM) or vehicle control for 48 hours prior to cell counting. Columns represent Mean ± SEM (N = 6). Student's t-test shGFP Vehicle vs. shGFP Torin 1 (P = 2.83 E-07), shGFP Vehicle vs. shArfGAP1 Vehicle (*P = 0.025), shGFP Torin 1 vs, shArfGAP1 Torin 1 (P = 0.139), and shArfGAP1 Vehicle vs. shArfGAP1 Torin 1 (P = 1.22 E-07). "ns" stands for "not significant".


Figure 1Tau levels in the cortex ( A , C - E ) and hippocampus ( B , F - H ) of 4 - 10 - month - old mice were determined by western blot analysis . ( A , B ) Representative western blots of cortical ( A ) and hippocampal ( B ) homogenates from mice of the indicated genotypes show levels of hTau ( HT7 ) and total tau ( Tau5 ) . Actin served as a loading control . ( C - H ) Quantitation of western blot signals for full - length tau detected with the mTau / hTau cross - reactive antibodies Tau5 ( C , F ) or EP2456Y ( D , G ) or the hTau - specific antibody Tau12 ( E , H ) . n = 3 - 8 mice per group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed unpaired t test ( E , H ) or by Welch ' s one - way ANOVA with post - hoc two - tailed Welch ' s t test ( C , D , F , G ) . P values were Holm - adjusted for multiple comparisons . Values are means ± SEM .

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Figure 1Tau levels in the cortex (A, C-E) and hippocampus (B, F-H) of 4-10-month-old mice were determined by western blot analysis. (A, B) Representative western blots of cortical (A) and hippocampal (B) homogenates from mice of the indicated genotypes show levels of hTau (HT7) and total tau (Tau5). Actin served as a loading control. (C-H) Quantitation of western blot signals for full-length tau detected with the mTau/hTau cross-reactive antibodies Tau5 (C, F) or EP2456Y (D, G) or the hTau-specific antibody Tau12 (E, H). n = 3-8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (E, H) or by Welch's one-way ANOVA with post-hoc two-tailed Welch's t test (C, D, F, G). P values were Holm-adjusted for multiple comparisons. Values are means ± SEM.


Figure 2Tau expression in the cortex ( A , C , E ) and hippocampus ( B , D , F ) of 4 - 10 - month - old mice was determined by quantitative RT - PCR ( A , B ) . ( A , B ) hTau mRNA levels in tissue homogenates were determined by quantitative RT - PCR . Tau expression in the cortex ( A , C , E ) and hippocampus ( B , D , F ) of 4 - 10 - month - old mice was determined by western blot analysis ( C - F ) . ( C , D ) Western blots from Figure 1A and B were enhanced to better reveal tau fragments of ~ 40 kDa . ( E , F ) Quantitation of western blot signals for tau fragments of ~ 40 kDa detected with Tau5 in tissue homogenates . n = 3 - 4 mice per group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed unpaired t test ( A , B ) or by Welch ' s one - way ANOVA with post - hoc two - tailed Welch ' s t test ( E , F ) . P values were Holm - adjusted . Values are means ± SEM .

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Figure 2Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4-10-month-old mice was determined by quantitative RT-PCR (A, B). (A, B) hTau mRNA levels in tissue homogenates were determined by quantitative RT-PCR.Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4-10-month-old mice was determined by western blot analysis (C-F).(C, D) Western blots from Figure 1A and B were enhanced to better reveal tau fragments of ~40 kDa. (E, F) Quantitation of western blot signals for tau fragments of ~40 kDa detected with Tau5 in tissue homogenates. n = 3-4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (A, B) or by Welch's one-way ANOVA with post-hoc two-tailed Welch's t test (E, F). P values were Holm-adjusted. Values are means ± SEM.


Figure 3Coronal brain sections from 2 - 4 - month - old mice of the indicated genotypes were immunostained with different tau antibodies . ( A - I ) Sections of hemibrain ( A - C ) , whole hippocampus ( D - F ) , and CA1 region ( G - I ) immunostained for hTau ( HT7 ) . ( J - O ) Hippocampal sections immunostained with phosphorylation - dependent tau antibodies : PHF1 ( p396 , 404 ) ( J - L ) , AT8 ( pSer 199 , 202 , and pThr 205 ) ( M - O ) . ( P - R ) Representative photomicrographs of GFAP immunostaining of the CA1 region ( P , Q ) and quantitation of GFAP immunoreactivity ( R ) .

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Figure 3Coronal brain sections from 2-4-month-old mice of the indicated genotypes were immunostained with different tau antibodies. (A-I) Sections of hemibrain (A-C), whole hippocampus (D-F), and CA1 region (G-I) immunostained for hTau (HT7). (J-O) Hippocampal sections immunostained with phosphorylation-dependent tau antibodies: PHF1 (p396, 404) (J-L), AT8 (pSer 199, 202, and pThr 205) (M-O).(P-R) Representative photomicrographs of GFAP immunostaining of the CA1 region (P, Q) and quantitation of GFAP immunoreactivity (R).


Figure 4Six - month - old hTau - A152T ( L1 ) mice and NTG controls were or were not treated with DOX for 2 months and analyzed at 8 months of age by immunohistochemistry . ( A - I ) Untreated hTau - A152T ( L1 ) mice had increased neuronal labeling with the hTau antibody HT7 ( A ) , the phosphorylation - dependent tau antibody PHF1 ( D ) , and the conformation - dependent tau antibody MC1 ( G ) . HT7 ( A ) and PHF1 ( D ) staining patterns were similar . MC1 ( G ) specifically stained hippocampal mossy fibers . In hTau - A152T ( L1 ) mice , DOX reduced hippocampal staining with HT7 , PHF1 , and MC1 ( B , E , H ) to the levels in DOX - treated NTG controls ( C , F , I ) . ( J - M ) DOX also normalized CA1 levels of GFAP immunoreactivity ( IR ) in hTau - A152T ( L1 ) mice , as shown by representative photomicrographs ( J - L ) and quantitation of GFAP immunoreactivity ( M ) . Scale bars : 300 µm ( A - I ) , 25 µm ( J - L ) . n = 3 - 4 mice per genotype . * P < 0 . 05 by two - tailed Welch ' s t test without Holm adjustment . Values are means ± SEM . a . u . , arbitrary units .

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Figure 4Six-month-old hTau-A152T(L1) mice and NTG controls were or were not treated with DOX for 2 months and analyzed at 8 months of age by immunohistochemistry. (A-I) Untreated hTau-A152T(L1) mice had increased neuronal labeling with the hTau antibody HT7 (A), the phosphorylation-dependent tau antibody PHF1 (D), and the conformation-dependent tau antibody MC1 (G). HT7 (A) and PHF1 (D) staining patterns were similar. MC1 (G) specifically stained hippocampal mossy fibers. In hTau-A152T(L1) mice, DOX reduced hippocampal staining with HT7, PHF1, and MC1 (B, E, H) to the levels in DOX-treated NTG controls (C, F, I).(J-M) DOX also normalized CA1 levels of GFAP immunoreactivity (IR) in hTau-A152T(L1) mice, as shown by representative photomicrographs (J-L) and quantitation of GFAP immunoreactivity (M). Scale bars: 300 µm (A-I), 25 µm (J-L). n = 3-4 mice per genotype. *P < 0.05 by two-tailed Welch's t test without Holm adjustment. Values are means ± SEM. a.u., arbitrary units.


Figure 5Nesting behavior of mice at 10 - 14 months of age was scored 0 - 7 by an investigator blinded to genotype . n = 17 NTG , 12 CaMKII - tTA , 13 hTau - WT ( L32 ) , and 10 hTau - A152T ( L1 ) . Nest building was significantly impaired only in hTau - A152T ( L1 ) mice ( P < 0 . 01 at 2 and 6 h ) and CaMKII - tTA mice ( P < 0 . 05 at 2 h , P < 0 . 01 at 6 hours ) by nonparametric Wilcoxon rank - sum test with gatekeeping approach and Holm adjustment . Values are means ± SEM .

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Figure 5Nesting behavior of mice at 10-14 months of age was scored 0-7 by an investigator blinded to genotype. n = 17 NTG, 12 CaMKII-tTA, 13 hTau-WT(L32), and 10 hTau-A152T(L1). Nest building was significantly impaired only in hTau-A152T(L1) mice (P < 0.01 at 2 and 6 h) and CaMKII-tTA mice (P < 0.05 at 2 h, P < 0.01 at 6 hours) by nonparametric Wilcoxon rank-sum test with gatekeeping approach and Holm adjustment. Values are means ± SEM.


Figure 6Mice from cohorts 1 - 3 were tested in the MWM at the indicated ages . ( A - D , M , N ) Learning curves of mice in cohorts 1 ( A - D ) and 3 ( M , N ) . Data at day 0 are from the first trial on day 1 . Data from cohort 2 are shown in EV5B - G . Compared with NTG controls , only hTau - A152T ( L1 ) mice showed significant learning impairments at 4 - 6 , 17 - 19 ( cohort 1 , P < 0 . 001 ) and 17 - 21 ( cohort 3 , P < 0 . 01 ) months of age by Cox proportional hazards model analysis with mixed effects and Holm adjustment for the following comparisons for each age range : NTG versus CaMKII - tTA , NTG versus TRE - hTau - A152T ( L1 ) , and NTG versus hTau - A152T ( L1 ) for cohort 1 , and NTG versus CaMKII - tTA , NTG versus hTau - WT ( L32 ) , NTG versus hTau - A152T ( L1 ) , and hTau - WT ( L32 ) versus hTau - A152T ( L1 ) for cohort 3 . ( E - L , O - V ) Performance of mice from cohorts 1 ( E - L ) , 3 ( O - R ) and 1 - 3 combined ( S - V ) in a probe trial ( platform removed ) 24 h after the last training trial . ( E - H , O , P , S , T ) Latency to reach original platform location . ( I - L , Q , R , U , V ) Number of times mice crossed the original platform location ( black bars ) or the average of their crossings of corresponding locations in the three other quadrants ( white bars ) . Cohort 1 : n = 12 ( A - L ) NTG ; 13 ( A , B , E , F , I , J ) , 11 ( C , G , K ) , or 9 ( D , H , L ) TRE - hTau - A152T ( L1 ) ; 11 ( A , B , E , F , I , J ) , 10 ( C , G , K ) , or 9 ( D , H , L ) CaMKII - tTA ; and 14 ( A , B , E , F , I , J ) , 13 ( C , G , K ) , or 12 ( D , H , L ) hTau - A152T ( L1 ) mice . Cohort 3 : n = 17 ( M - R ) NTG ; 12 ( M , O , Q ) or 11 ( N , P , R ) CaMKII - tTA ; 13 ( M , O , Q ) or 12 ( N , P , R ) hTau - WT ( L32 ) ; and 10 ( M - R ) hTau - A152T ( L1 ) mice . Cohorts 1 + 2 + 3 : n = 47 ( S , U ) , or 39 ( T , V ) NTG ; 11 ( S , U ) or 9 ( T , V ) TRE - hTau - A152T ( L1 ) ; 34 ( S , U ) or 31 ( T , V ) CaMKII - tTA ; 13 ( S , U ) or 12 ( T , V ) hTau - WT ( L32 ) ; and 43 ( S , U ) or 36 ( T , V ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Cox proportional hazards / mixed effects model analysis ( A - D , M , N ) , one - way ANOVA with post - hoc Tukey ( E - H , O , P ) or Dunnett ' s ( S , T ) test , or one - tailed paired t test with Holm adjustment ( I - L , Q , R , U , V ) . Values are means ± SEM .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6Mice from cohorts 1-3 were tested in the MWM at the indicated ages. (A-D, M, N) Learning curves of mice in cohorts 1 (A-D) and 3 (M, N). Data at day 0 are from the first trial on day 1. Data from cohort 2 are shown in EV5B-G. Compared with NTG controls, only hTau-A152T(L1) mice showed significant learning impairments at 4-6, 17-19 (cohort 1, P < 0.001) and 17-21 (cohort 3, P < 0.01) months of age by Cox proportional hazards model analysis with mixed effects and Holm adjustment for the following comparisons for each age range: NTG versus CaMKII-tTA, NTG versus TRE-hTau-A152T(L1), and NTG versus hTau-A152T(L1) for cohort 1, and NTG versus CaMKII-tTA, NTG versus hTau-WT(L32), NTG versus hTau-A152T(L1), and hTau-WT(L32) versus hTau-A152T(L1) for cohort 3. (E-L, O-V) Performance of mice from cohorts 1 (E-L), 3 (O-R) and 1-3 combined (S-V) in a probe trial (platform removed) 24 h after the last training trial. (E-H, O, P, S, T) Latency to reach original platform location. (I-L, Q, R, U, V) Number of times mice crossed the original platform location (black bars) or the average of their crossings of corresponding locations in the three other quadrants (white bars). Cohort 1: n = 12 (A-L) NTG; 13 (A, B, E, F, I, J), 11 (C, G, K), or 9 (D, H, L) TRE-hTau-A152T(L1); 11 (A, B, E, F, I, J), 10 (C, G, K), or 9 (D, H, L) CaMKII-tTA; and 14 (A, B, E, F, I, J), 13 (C, G, K), or 12 (D, H, L) hTau-A152T(L1) mice. Cohort 3: n = 17 (M-R) NTG; 12 (M, O, Q) or 11 (N, P, R) CaMKII-tTA; 13 (M, O, Q) or 12 (N, P, R) hTau-WT(L32); and 10 (M-R) hTau-A152T(L1) mice. Cohorts 1+2+3: n = 47 (S, U), or 39 (T, V) NTG; 11 (S, U) or 9 (T, V) TRE-hTau-A152T(L1); 34 (S, U) or 31 (T, V) CaMKII-tTA; 13 (S, U) or 12 (T, V) hTau-WT(L32); and 43 (S, U) or 36 (T, V) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Cox proportional hazards/mixed effects model analysis (A-D, M, N), one-way ANOVA with post-hoc Tukey (E-H, O, P) or Dunnett's (S, T) test, or one-tailed paired t test with Holm adjustment (I-L, Q, R, U, V). Values are means ± SEM.


Figure 7 ( A - C ) One cohort of mice was assessed with the social approach test at young ( A ) and old ( B ) age and another cohort of mice at middle age ( C ) . Mice were placed individually into the empty center chamber of a three - chamber apparatus . One side - chamber contained an empty cup , and the other an identical cup with an unfamiliar live mouse in it . Close contact with each cup was monitored for 10 min . n = 12 ( A , B ) or 17 ( C ) NTG ; 13 ( A ) or 10 ( B ) TRE - hTau - A152T ( L1 ) ; 12 ( A ) , 9 ( B ) , or 12 ( C ) CamKII - tTA ; 13 ( C ) hTau - WT ( L32 ) ; and 14 ( A ) , 12 ( B ) , or 10 ( C ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - tailed paired t test with Holm adjustment . Values are means ± SEM .

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

Figure 7(A-C) One cohort of mice was assessed with the social approach test at young (A) and old (B) age and another cohort of mice at middle age (C). Mice were placed individually into the empty center chamber of a three-chamber apparatus. One side-chamber contained an empty cup, and the other an identical cup with an unfamiliar live mouse in it. Close contact with each cup was monitored for 10 min. n = 12 (A, B) or 17 (C) NTG; 13 (A) or 10 (B) TRE-hTau-A152T(L1); 12 (A), 9 (B), or 12 (C) CamKII-tTA; 13 (C) hTau-WT(L32); and 14 (A), 12 (B), or 10 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-tailed paired t test with Holm adjustment. Values are means ± SEM.


Figure 8 ( A - F ) Coronal brain sections obtained from mice at 4 - 6 ( A - C ) or 20 - 23 ( D - F ) months of age were immunostained for NeuN . The number of NeuN - positive cells with neuronal morphology was determined in dentate gyrus ( A , D ) , CA3 ( B , E ) , and CA1 ( C , F ) . n = 11 ( A - C ) or 18 ( D - F ) NTG ; 11 ( A - C ) or 5 ( D - F ) CamKII - tTA ; 7 ( A - C ) or 9 ( D - F ) hTau - WT ( L32 ) ; and 14 ( A - C ) or 10 ( D - F ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - way ANOVA and Tukey test . Values are means ± SEM .

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

Figure 8(A-F) Coronal brain sections obtained from mice at 4-6 (A-C) or 20-23 (D-F) months of age were immunostained for NeuN. The number of NeuN-positive cells with neuronal morphology was determined in dentate gyrus (A, D), CA3 (B, E), and CA1 (C, F). n = 11 (A-C) or 18 (D-F) NTG; 11 (A-C) or 5 (D-F) CamKII-tTA; 7 (A-C) or 9 (D-F) hTau-WT(L32); and 14 (A-C) or 10 (D-F) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Tukey test. Values are means ± SEM.


Figure 9fEPSPs in CA3 stratum lucidum were induced by stimulation of mossy fibers in acute hippocampal slices . ( A ) Representative fEPSP traces from slices of 4 - 8 - month - old mice . ( B , C ) Input / output curves reflecting the change in the fEPSP slope as a function of the fiber volley amplitude from slices obtained at 4 - 8 ( B ) or 20 ( C ) months of age . ( D , E ) Short - term plasticity assessed by measuring the PPR ( second pulse / first pulse ) with 50 - msec interpulse interval at 4 - 8 ( D ) or 20 ( E ) months of age . ( F ) Mossy fiber LTP at 20 months of age . n ( mice / slices ) = 5 - 9 / 8 - 21 ( NTG ) , 2 - 5 / 5 - 12 ( CaMKII - tTA ) , 3 - 6 / 8 - 14 ( TRE - hTau ) , 3 - 6 / 8 - 14 ( hTau - WT ) , and 5 - 6 / 8 - 14 ( hTau - A152T ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus NTG by linear regression analysis with Holm adjustment ( B , C ) or by one - way ANOVA and Dunnett ' s test ( D , E ) . No significant difference was detected by two - way ANOVA in ( F ) . Values are means ± SEM .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9fEPSPs in CA3 stratum lucidum were induced by stimulation of mossy fibers in acute hippocampal slices. (A) Representative fEPSP traces from slices of 4-8-month-old mice. (B, C) Input/output curves reflecting the change in the fEPSP slope as a function of the fiber volley amplitude from slices obtained at 4-8 (B) or 20 (C) months of age. (D, E) Short-term plasticity assessed by measuring the PPR (second pulse/first pulse) with 50-msec interpulse interval at 4-8 (D) or 20 (E) months of age.(F) Mossy fiber LTP at 20 months of age. n (mice/slices) = 5-9/8-21 (NTG), 2-5/5-12 (CaMKII-tTA), 3-6/8-14 (TRE-hTau), 3-6/8-14 (hTau-WT), and 5-6/8-14 (hTau-A152T). *P < 0.05, **P < 0.01, ***P < 0.001 versus NTG by linear regression analysis with Holm adjustment (B, C) or by one-way ANOVA and Dunnett's test (D, E). No significant difference was detected by two-way ANOVA in (F). Values are means ± SEM.


Figure 10 ( A - C ) Subdural EEG recordings from freely behaving mice at 4 - 9 months of age before ( A top trace , B ) and after ( A bottom trace , C ) injection of PTZ at a dose ( 30 mg / kg ) that did not produce convulsions . ( A ) Representative EEG traces from an hTau - A152T ( L1 ) mouse . Arrows indicate epileptic discharges . ( B , C ) Quantitation of spikes per hour at baseline ( B ) and of spikes per 20 - min intervals after PTZ injection ( C ) . n = 31 ( B ) or 23 ( C ) NTG ; 13 ( B ) or 11 ( C ) CaMKII - tTA ; 12 ( B ) or 10 ( C ) hTau - WT ( L32 ) ; and 22 ( B ) or 21 ( C ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Welch ' s one - way ANOVA and two - tailed Welch ' s t test with Holm adjustment ( B ) or by two - way repeated - measures ANOVA and Tukey test ( C ) . Values are means ± SEM .

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

Figure 10(A-C) Subdural EEG recordings from freely behaving mice at 4-9 months of age before (A top trace, B) and after (A bottom trace, C) injection of PTZ at a dose (30 mg/kg) that did not produce convulsions. (A) Representative EEG traces from an hTau-A152T(L1) mouse. Arrows indicate epileptic discharges. (B, C) Quantitation of spikes per hour at baseline (B) and of spikes per 20-min intervals after PTZ injection (C). n = 31 (B) or 23 (C) NTG; 13 (B) or 11 (C) CaMKII-tTA; 12 (B) or 10 (C) hTau-WT(L32); and 22 (B) or 21 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch's one-way ANOVA and two-tailed Welch's t test with Holm adjustment (B) or by two-way repeated-measures ANOVA and Tukey test (C). Values are means ± SEM.


Figure 11 ( A - D ) Numbers of offspring from crosses of hAPP - J20 mice with hTau - A152T ( L1 ) ( A , B ) or hTau - WT ( L32 ) ( C , D ) mice that were ( A , C ) or were not ( B , D ) treated with DOX . ( E ) Numbers of offspring from crosses of lower expresser hAPP - J9 mice with hTau - A152T ( L1 ) mice that were not treated with DOX . n = 141 ( A ) , 112 ( B ) , 94 ( C ) , 83 ( D ) , and 282 ( E ) mice per cohort . Yields from two crosses indicated significant deviations from Mendelian inheritance : hAPP - J20 x TRE - hTau - A152T ( L1 ) x CaMKII - tTA without DOX ( Holm - adjusted P = 0 . 003 , unadjusted P = 0 . 0008 ) and hAPP - J9 x TRE - hTau - WT ( L32 ) x CaMKII - tTA without DOX ( adjusted and unadjusted P < 0 . 0001 ) by chi - square goodness - of - fit test . ( F ) Subdural EEG recordings from untreated 3 - 10 - month - old mice were analyzed to compare their number of spikes at baseline . n = 8 NTG , 6 hAPP - J9 , 6 hTau - A152T ( L1 ) , and 8 hAPP - J9 / hTau - A152T ( L1 ) . * P < 0 . 05 , * * P < 0 . 01 by logistic regression analysis for three - way interaction of the three transgenes with Holm adjustment ( A - E ) or by two - tailed Welch ' s t test with Holm adjustment ( F ) .

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 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 11(A-D) Numbers of offspring from crosses of hAPP-J20 mice with hTau-A152T(L1) (A, B) or hTau-WT(L32) (C, D) mice that were (A, C) or were not (B, D) treated with DOX. (E) Numbers of offspring from crosses of lower expresser hAPP-J9 mice with hTau-A152T(L1) mice that were not treated with DOX. n = 141 (A), 112 (B), 94 (C), 83 (D), and 282 (E) mice per cohort. Yields from two crosses indicated significant deviations from Mendelian inheritance: hAPP-J20 x TRE-hTau-A152T(L1) x CaMKII-tTA without DOX (Holm-adjusted P = 0.003, unadjusted P = 0.0008) and hAPP-J9 x TRE-hTau-WT(L32) x CaMKII-tTA without DOX (adjusted and unadjusted P < 0.0001) by chi-square goodness-of-fit test.(F) Subdural EEG recordings from untreated 3-10-month-old mice were analyzed to compare their number of spikes at baseline. n = 8 NTG, 6 hAPP-J9, 6 hTau-A152T(L1), and 8 hAPP-J9/hTau-A152T(L1). *P < 0.05, **P < 0.01 by logistic regression analysis for three-way interaction of the three transgenes with Holm adjustment (A-E) or by two-tailed Welch's t test with Holm adjustment (F).


Figure 2Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA _ KO mutants versus WT . Of 759 predicted miRNA target genes , 728 ( 96 % ) were detectable in the ribosome profiling data and appear in the plot . Data information : In A , student ' s t - test was applied to assess the significance for the increase in ribosome occupancy of the 728 / 759 miRNA targets ( p - value < 1e51 for every mutant ) . Top : Immunoblot analysis of CIC in WT , Drosha _ KO , Dicer _ KO and Ago2 & 1 _ KO mESCs . TUBULIN was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing quantification of CIC intensity , normalized to TUBULIN and relative to the WT sample in three biological replicates . Data information : Bar graph in Fig 2F shows mean intensity of CIC signal ± SD normalized to TUBULIN . Values are relative to WT , which was set to 1 . P - values were calculated using a student ' s t - test comparing each value to the WT . * p - value < 0 . 05 .

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 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 2Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA_KO mutants versus WT. Of 759 predicted miRNA target genes, 728 (96%) were detectable in the ribosome profiling data and appear in the plot. Data information: In A, student's t-test was applied to assess the significance for the increase in ribosome occupancy of the 728/759 miRNA targets (p-value<1e51 for every mutant).Top: Immunoblot analysis of CIC in WT, Drosha_KO, Dicer_KO and Ago2&1_KO mESCs. TUBULIN was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to TUBULIN and relative to the WT sample in three biological replicates. Data information: Bar graph in Fig 2F shows mean intensity of CIC signal ± SD normalized to TUBULIN. Values are relative to WT, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05.


Figure 3MA plot of the DGE analysis in miR - 290 - 295 _ KO mESCs . Data information : In A , significant genes in MA plot were determined using an adjusted p - value of 0 . 1 . F , G . Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR - 290 - 295 . In addition to the data shown and described in Fig 1D , E , a second AGO2 - binding dataset , based on cross - linking immunoprecipitation and sequencing ( CLIP - seq ) , has been integrated . Response elements for miR - 290 - 295 cluster members are denoted in red and labeled . Top : Immunoblot analysis of TFAP4 in WT , Drosha _ KO , Dicer _ KO , Ago2 & 1 _ KO and miR - 290 - 295 _ KO mESCs . LAMINB1 was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing quantification of CIC intensity , normalized to LAMINB1 and relative to the WT sample in three biological replicates . Data information : In H bar graphs show mean intensity of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or the negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - test comparing each value to the WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 . Top : Immunoblot analysis of TFAP4 after transfection of miRNA mimics ( miR - 291a - 5p , miR - 291a - 3p and miR - 291a - 5p + miR - 291a - 3p combined ) in miR - 290 - 295 _ KO mESCs . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing quantification of TFAP4 intensity , normalized to Coomassie and relative to the WT sample in three biological replicates . Data information : In I , bar graphs show mean intensity of TFAP4 signal ± SD normalized to Coomassie . Values are relative to WT or the negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - test comparing each value to the WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 . Top : Mature sequences of 5p and 3p miRNAs of the miR - 290 - 295 family and the mature sequence of the human miR - 302c - 3p . Colors indicate identical seed sequences . Bottom : AGO2 CLIP - seq shows a peak at the miR - 302c - 3p binding site on human TFAP4 .

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 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 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 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 3MA plot of the DGE analysis in miR-290-295_KO mESCs. Data information: In A, significant genes in MA plot were determined using an adjusted p-value of 0.1.F, G. Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR-290-295. In addition to the data shown and described in Fig 1D, E, a second AGO2-binding dataset, based on cross-linking immunoprecipitation and sequencing (CLIP-seq), has been integrated. Response elements for miR-290-295 cluster members are denoted in red and labeled.Top: Immunoblot analysis of TFAP4 in WT, Drosha_KO, Dicer_KO, Ago2&1_KO and miR-290-295_KO mESCs. LAMINB1 was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to LAMINB1 and relative to the WT sample in three biological replicates. Data information: In H bar graphs show mean intensity of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.Top: Immunoblot analysis of TFAP4 after transfection of miRNA mimics (miR-291a-5p, miR-291a-3p and miR-291a-5p+miR-291a-3p combined) in miR-290-295_KO mESCs. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of TFAP4 intensity, normalized to Coomassie and relative to the WT sample in three biological replicates. Data information: In I, bar graphs show mean intensity of TFAP4 signal ± SD normalized to Coomassie. Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.Top: Mature sequences of 5p and 3p miRNAs of the miR-290-295 family and the mature sequence of the human miR-302c-3p. Colors indicate identical seed sequences. Bottom: AGO2 CLIP-seq shows a peak at the miR-302c-3p binding site on human TFAP4.


Figure 4Immunoblot validation of siPOOL - mediated knock down of Tfap4 . TFAP4 levels were compared between untreated WT versus miR - 290 - 295 _ KO cells treated with a negative control and a Tfap4 - targeted siPOOL . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of two biological replicates . Scatterplot of DEG in miR - 290 - 295 _ KO control versus siPOOL - Tfap4 . SiPOOL experiments were performed in miR - 290 - 295 _ KO cells and DE was assessed relative to a negative control siPOOL transfection . Only genes that are statistically significantly differentially expressed in miR - 290 - 295 _ KO are shown . Genes predicted to be targeted by miR - 290 - 295 are marked in blue . Genes are defined as rescued ( orange ) if the log2FoldChange - ratio between miR - 290 - 295 _ KO control and siPOOL - Tfap4 is in the range [ - 2 , - 1 / 2 ] . Tfap4 is marked by a red dot . Representative images of NANOG ( red ) and OCT4 ( green ) immunofluorescence staining in WT , Drosha _ KO and Drosha & Tfap4 _ KO mESCs . Scale bar = 20 μm . Quantification of NANOG fluorescence signal intensity in WT , Drosha _ KO and Drosha & Tfap4 _ KO mESCs show no significant rescue of NANOG intensity in Drosha & Tfap4 _ KO Data information : In E , box plots show nuclear intensity of NANOG in ~ 300 cells per genotype . P - values were generated using ordinary one - way ANOVA . * * * * p - value < 0 . 0001 . In panel E , the box plot ' s central band represents the median relative intensity for each genotype . The boxes represent the interquartile range and the whiskers represent the maximum and minimum relative intensity values .

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Figure 4Immunoblot validation of siPOOL-mediated knock down of Tfap4. TFAP4 levels were compared between untreated WT versus miR-290-295_KO cells treated with a negative control and a Tfap4-targeted siPOOL. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of two biological replicates.Scatterplot of DEG in miR-290-295_KO control versus siPOOL-Tfap4. SiPOOL experiments were performed in miR-290-295_KO cells and DE was assessed relative to a negative control siPOOL transfection. Only genes that are statistically significantly differentially expressed in miR-290-295_KO are shown. Genes predicted to be targeted by miR-290-295 are marked in blue. Genes are defined as rescued (orange) if the log2FoldChange-ratio between miR-290-295_KO control and siPOOL-Tfap4 is in the range [-2, -1/2]. Tfap4 is marked by a red dot.Representative images of NANOG (red) and OCT4 (green) immunofluorescence staining in WT, Drosha_KO and Drosha & Tfap4_KO mESCs. Scale bar = 20 μm. Quantification of NANOG fluorescence signal intensity in WT, Drosha_KO and Drosha & Tfap4_KO mESCs show no significant rescue of NANOG intensity in Drosha & Tfap4_KO Data information: In E, box plots show nuclear intensity of NANOG in ~300 cells per genotype. P-values were generated using ordinary one-way ANOVA. ****p-value<0.0001. In panel E, the box plot's central band represents the median relative intensity for each genotype. The boxes represent the interquartile range and the whiskers represent the maximum and minimum relative intensity values.


Figure 1Confocal images of mPS - PI stained 6 - day - old seedlings of Col - 0 ( A ) , bravo - 2 ( B ) , wox5 - 1 ( C ) and bravo - 2 wox5 - 1 ( D ) mutants . Left black arrows indicate QC cells and right white arrows indicate CSC . Scale bar : 50 µm . Quantification of the QC divisions in 6 - day - old roots expressed in percentage ( n > 50 , 3 replicates ) . D : QC divided ; ND : QC non divided . Quantification of CSC layers in 6 - day - old roots expressed in percentage ( n > 50 , 3 replicates ) . Lateral root density ( number of lateral roots per mm of root length ) of 7 - day - olf WT , bravo - 2 , wox5 - 1 and bravo - 2 wox5 - 1 mutants ( n > 40 , 2 replicates ) . Different letters indicate statistically significant differences ( p - value < 0 . 05 Student ´ s t - test ) . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers .

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Figure 1Confocal images of mPS-PI stained 6-day-old seedlings of Col-0 (A), bravo-2 (B), wox5-1 (C) and bravo-2 wox5-1 (D) mutants. Left black arrows indicate QC cells and right white arrows indicate CSC. Scale bar: 50 µm.Quantification of the QC divisions in 6-day-old roots expressed in percentage (n>50, 3 replicates). D: QC divided; ND: QC non divided.Quantification of CSC layers in 6-day-old roots expressed in percentage (n>50, 3 replicates).Lateral root density (number of lateral roots per mm of root length) of 7-day-olf WT, bravo-2, wox5-1 and bravo-2 wox5-1 mutants (n>40, 2 replicates). Different letters indicate statistically significant differences (p- value < 0.05 Student´s t-test). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.


Figure 2Confocal images of PI - stained 6 - day - old roots . GFP - tagged expression is shown in yellow . A - C ) pBRAVO : GFP in WT ( A ) , bravo - 2 ( B ) and wox5 - 1 ( C ) knockout backgrounds . D - G ) pWOX5 : GFP in the WT ( D ) , bravo - 2 ( E ) , wox5 - 1 ( F ) and bravo - 2 wox5 - 1 ( G ) knockout backgrounds . Scale bar : 50 µm . Quantification of the GFP fluorescent signal of the roots in A - C ( H ) and D - G ( I ) . Boxplot indicating the average pixel intensity of the GFP in the stem cell niche ( n > 25 , 3 biological replicates , * p - value < 0 . 05 Student ´ s t - test for each genotype versus the WT in the same condition ) . Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN ( Appendix Figure S7 ) . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers and black dots are the experimental observations .

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Figure 2Confocal images of PI-stained 6-day-old roots. GFP-tagged expression is shown in yellow. A-C) pBRAVO:GFP in WT (A), bravo-2 (B) and wox5-1 (C) knockout backgrounds. D-G) pWOX5:GFP in the WT (D), bravo-2 (E), wox5-1 (F) and bravo-2 wox5-1 (G) knockout backgrounds. Scale bar: 50 µm. Quantification of the GFP fluorescent signal of the roots in A-C (H) and D-G (I). Boxplot indicating the average pixel intensity of the GFP in the stem cell niche (n>25, 3 biological replicates, *p-value < 0.05 Student´s t- test for each genotype versus the WT in the same condition). Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN (Appendix Figure S7). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers and black dots are the experimental observations.


Figure 3Parameter space exploration of ( C ) the alleviation and the ( D ) activation models . Boxplots showing the distribution of stationary promoter pBRAVO ( pB ) and pWOX5 ( pW ) fold - changes in each mutant and overexpression lines over the WT obtained for different parameter values . Superindexes denote the genotype , standing bravo for bravo mutant , wox5 for wox5 mutant , dm for the bravo wox5 double mutant , Woe for WOX5 overexpression and Boe for BRAVO overexpression . Box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . The results are obtained by solving N = 1000 different runs of each model at the stationary state ( red and blue stripplots ) for the WT , mutants and overexpressor scenarios . These N runs differ in the parameter values , which are chosen at random from a uniform distribution

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Figure 3Parameter space exploration of (C) the alleviation and the (D) activation models. Boxplots showing the distribution of stationary promoter pBRAVO (pB) and pWOX5 (pW) fold-changes in each mutant and overexpression lines over the WT obtained for different parameter values. Superindexes denote the genotype, standing bravo for bravo mutant, wox5 for wox5 mutant, dm for the bravo wox5 double mutant, Woe for WOX5 overexpression and Boe for BRAVO overexpression. Box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. The results are obtained by solving N=1000 different runs of each model at the stationary state (red and blue stripplots) for the WT, mutants and overexpressor scenarios. These N runs differ in the parameter values, which are chosen at random from a uniform distribution between


Figure 4Interaction of BRAVO with WOX5 ( B ) , BES1 ( C ) , BES1D ( D ) and TPL ( E ) ; and interaction of WOX5 with BRAVO ( G ) , BES1 ( H ) , BES1D ( I ) and TPL ( J ) measured by FRET - FLIM . GFP fluorescence lifetime τ [ ns ] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells . GFP fluorescence lifetime fitted pixel - wise with a mono - exponential model of BRAVO and WOX5 interactions . mV , mVenus ; mCh , mCherry . Scale bar : 5 µm . Fluorescence - weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double - exponential model of the indicated samples are summarized in box plots . Statistical significance was tested by one - way ANOVA with a Sidakholm post - hoc test . Different letters indicate statistically significant differences ( p - value < 0 . 01 ) . For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers . Yeast two - hybrid assay showing BRAVO interacting with WOX5 , BES1 - D and TPL ; and BES1 - D interacting with TPL . In the left column yeast cells were grown on control media , and in the right column yeast cells were grown on control media lacking Leu , Trp and His , indicating an interaction between the proteins .

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Figure 4Interaction of BRAVO with WOX5 (B), BES1 (C), BES1D (D) and TPL (E); and interaction of WOX5 with BRAVO (G), BES1 (H), BES1D (I) and TPL (J) measured by FRET-FLIM. GFP fluorescence lifetime τ [ns] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells. GFP fluorescence lifetime fitted pixel-wise with a mono-exponential model of BRAVO and WOX5 interactions. mV, mVenus; mCh, mCherry. Scale bar: 5 µm.Fluorescence-weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double-exponential model of the indicated samples are summarized in box plots. Statistical significance was tested by one-way ANOVA with a Sidakholm post-hoc test. Different letters indicate statistically significant differences (p-value < 0.01). For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured. In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.Yeast two-hybrid assay showing BRAVO interacting with WOX5, BES1-D and TPL; and BES1-D interacting with TPL. In the left column yeast cells were grown on control media, and in the right column yeast cells were grown on control media lacking Leu, Trp and His, indicating an interaction between the proteins.


Figure 6Expression of the 380 common BRAVO and WOX5 regulated genes from ( B ) . Expression of the 53 genes from ( C ) showing similar expression in bravo and wox5 mutants . Expression of the 41 genes from ( C ) showing opposite expression levels in bravo and wox5 mutants .

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Figure 6Expression of the 380 common BRAVO and WOX5 regulated genes from (B). Expression of the 53 genes from (C) showing similar expression in bravo and wox5 mutants. Expression of the 41 genes from (C) showing opposite expression levels in bravo and wox5 mutants.


Figure 1LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) and cell lysates ( Lysate ) were collected and immunoblotted with the indicated antibodies （ A ） . LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected and ( B - E ) IL - 1β secretion ( B ) , IL - 18 secretion ( C ) and TNF - ɑ secretion ( D ) and LDH release ( E ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments . LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . ( F ) HBVLV - Vdr expression in Vdr - / - BMDMs by lentivirus - mediated transduction primed with LPS and infected with 50 MOI S . Typhimurium for 2 h , cell lysates , and culture supernatants ( SN ) were collected and immunoblotted with the indicated antibodies . ( G ) LPS - primed WT and Vdr - / - BMDMs were treated with 1 µg / ml FLIC ( LFn - Flagellin and PA ) or PrgJ （ 1 µg / ml LFn - PrgJ and PA ） for 1 h , total mixtures ( culture supernatants and cell lysates ) were collected and immunoblotted with the indicated antibodies . ( H ) LPS - primed WT , Vdr - / - , NLRC4 - / - and Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) and cell lysates were collected and immunoblotted with the indicated antibodies . LPS - primed WT , Vdr - / - , NLRC4 - / - and Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) were collected and ( I - L ) IL - 1β secretion ( I ) , IL - 18 secretion ( J ) and TNF - ɑ secretion ( K ) and LDH release ( L ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments .

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Figure 1LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) and cell lysates(Lysate) were collected and immunoblotted with the indicated antibodies（A）.LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) were collected and (B-E) IL-1β secretion(B), IL-18 secretion (C) and TNF-ɑ secretion (D) and LDH release(E) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. (F) HBVLV-Vdr expression in Vdr-/- BMDMs by lentivirus-mediated transduction primed with LPS and infected with 50 MOI S. Typhimurium for 2 h, cell lysates, and culture supernatants (SN) were collected and immunoblotted with the indicated antibodies.(G) LPS-primed WT and Vdr-/- BMDMs were treated with 1 µg/ml FLIC (LFn-Flagellin and PA) or PrgJ （1 µg/ml LFn- PrgJ and PA）for 1 h, total mixtures( culture supernatants and cell lysates) were collected and immunoblotted with the indicated antibodies.(H) LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) and cell lysates were collected and immunoblotted with the indicated antibodies.LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) were collected and (I-L) IL-1β secretion(I), IL-18 secretion (J) and TNF-ɑ secretion (K) and LDH release(L) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.


Figure 3LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) and Cell lysates were collected and immunoblotted with the indicated antibodies ( A ) . ELISALPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected antibodies ( A ) . ELISA detected TNF - ɑ ( B ) andIL - 1β ( C ) in supernatants . Data information : n ≥ 3 biological replicates . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 . LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with S . Typhimurium Bacterial burden was detected in spleens ( D ) , PCF ( E ) , and blood ( F ) of WT , Vdr - / - , NLRP3 - / - , and NLRP3 - / - Vdr - / - mice 24 h after S . Typhimurium infection . Data information : n ≥ 3 biological replicates . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 .

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Figure 3LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) and Cell lysates were collected and immunoblotted with the indicated antibodies(A).ELISALPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) were collected antibodies(A).ELISA detected TNF-ɑ(B)andIL-1β (C) in supernatants. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with S. Typhimurium Bacterial burden was detected in spleens(D), PCF(E), and blood (F)of WT, Vdr-/-, NLRP3-/-, and NLRP3-/- Vdr-/- mice 24 h after S. Typhimurium infection. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.


Figure 4A LPS - primed WT and Vdr - / - BMDMs were infected with S . Typhimurium at an MOI of 50 for 2 h . Endogenous ASC specks ( arrows ) were represented and quantified by immunofluorescence images . The data show representative results from three independent experiments : scale bar , 20 μm . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * * , p < 0 . 001 . B ASC oligomerization induced by the indicated stimuli in WT and Vdr − / − BMDMs primed with LPS .

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Figure 4A LPS-primed WT and Vdr-/- BMDMs were infected with S. Typhimurium at an MOI of 50 for 2 h. Endogenous ASC specks (arrows) were represented and quantified by immunofluorescence images. The data show representative results from three independent experiments: scale bar, 20 μm. Data are presented as the mean ± SEM; determined by Student's t-test ; ***,p < 0.001.B ASC oligomerization induced by the indicated stimuli in WT and Vdr −/− BMDMs primed with LPS.


Figure 5LPS - primed BMDMs were infected with S . Typhimurium at an MOI of 50 for 2h . Lysates were immunoprecipitated ( IP ) using an anti - VDR antibody and were immunoblotted with the indicated antibodies ( A ) . Whole - cell lysates were shown as the input . Data are representative of three independent experiments . ( B - C ) Flag - VDR was co - expressed with Myc - NLRC4 in HEK293T cells ; proteins were immunoprecipitated and analyzed by immunoblotting with indicated antibodies . Whole - cell lysates were shown as the input . Data are representative of three independent experiments . ( D ) Purified GST - VDR was incubated with purified His - NLRC4 for 2 h . His - NLRC4 - bound to GST - VDR was pulled down by glutathione beads and subjected to immunoblot analysis . Data are representative of three independent experiments . ( E ) WT or mutant NLRC4 ( CARD , NACHT , or LRR ( leucine - rich repeat ) ) and HA - VDR were expressed in HEK293T cells , immunoprecipitated , and analyzed by immunoblotting with indicated antibodies . * no specific band . Data are representative of three independent experiments . ( F ) WT or mutant VDR ( DBD or LBD ) and Myc - NLRC4 were expressed in HEK293T cells , immunoprecipitated , and analyzed by immunoblotting with indicated antibodies . Data are representative of three independent experiments .

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Figure 5LPS-primed BMDMs were infected with S. Typhimurium at an MOI of 50 for 2h. Lysates were immunoprecipitated (IP) using an anti-VDR antibody and were immunoblotted with the indicated antibodies(A). Whole-cell lysates were shown as the input. Data are representative of three independent experiments.(B-C) Flag-VDR was co-expressed with Myc-NLRC4 in HEK293T cells; proteins were immunoprecipitated and analyzed by immunoblotting with indicated antibodies. Whole-cell lysates were shown as the input. Data are representative of three independent experiments.(D) Purified GST-VDR was incubated with purified His-NLRC4 for 2 h. His-NLRC4- bound to GST-VDR was pulled down by glutathione beads and subjected to immunoblot analysis. Data are representative of three independent experiments.(E) WT or mutant NLRC4 (CARD, NACHT, or LRR (leucine-rich repeat)) and HA-VDR were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies.*no specific band. Data are representative of three independent experiments.(F) WT or mutant VDR (DBD or LBD) and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies. Data are representative of three independent experiments.


Figure 6A - B HEK293T cells were transfected with Flag - NLRC4 , VDR , Myc - FLagellin , and HA - NAIP5 ( A ) or Flag - NLRC4 , VDR , Myc - PrgJ and HA - NAIP2 ( B ) Samples were immunoprecipitated with the anti - Flag and analyzed by immunoblotting with indicated antibodies . HEK293T cells were transfected with Flag - NLRC4 , LBD - VDR , Myc - FLagellin , and HA - NAIP5 ( C ) or Flag - NLRC4 , LBD - VDR , Myc - PrgJ , and HA - NAIP2 ( D ) . Samples were immunoprecipitated with the anti - Flag and analyzed by immunoblotting with indicated antibodies .

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Figure 6A-B HEK293T cells were transfected with Flag-NLRC4, VDR, Myc-FLagellin, and HA-NAIP5 (A) or Flag-NLRC4, VDR, Myc- PrgJ and HA-NAIP2(B) Samples were immunoprecipitated with the anti-Flag and analyzed by immunoblotting with indicated antibodies.HEK293T cells were transfected with Flag-NLRC4, LBD-VDR, Myc-FLagellin, and HA-NAIP5 (C) or Flag-NLRC4, LBD-VDR, Myc-PrgJ, and HA-NAIP2 (D). Samples were immunoprecipitated with the anti-Flag and analyzed by immunoblotting with indicated antibodies.


Figure 7B VDR or mutant VDR ( K123A ) and Myc - NLRC4 were expressed in HEK293T cells , immunoprecipitated with the anti - Flag antibody , and analyzed by immunoblotting with indicated antibodies . C Transfected VDR or mutant VDR ( K123A ) and Flag - NLRC4 , Myc - PrgJ , and HA - NAIP2 in HEK293T cells , immunoprecipitated with the anti - HA antibody , and analyzed by immunoblotting with indicated antibodies . D Vdr - / - BMDMs were infected with lentivirus , which expressed Vector ( NC ) , VDR , and VDR - K123A , infected with S . Typhimurium at an MOI of 50 for 2 h , cell lysates ( Lysate ) and culture supernatants ( SN ) were collected and immunoblotted with the indicated antibodies . E - H LDH release ( E ) , IL - 1β secretion ( F ) , IL - 18 secretion ( G ) and TNF - ɑ secretion ( H ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; In each panel , data are representative of at least three independent experiments .

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Figure 7B VDR or mutant VDR(K123A)and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated with the anti-Flag antibody, and analyzed by immunoblotting with indicated antibodies.C Transfected VDR or mutant VDR(K123A) and Flag-NLRC4, Myc- PrgJ, and HA-NAIP2 in HEK293T cells, immunoprecipitated with the anti-HA antibody, and analyzed by immunoblotting with indicated antibodies.D Vdr-/- BMDMs were infected with lentivirus, which expressed Vector(NC), VDR, and VDR-K123A, infected with S. Typhimurium at an MOI of 50 for 2 h, cell lysates(Lysate) and culture supernatants(SN) were collected and immunoblotted with the indicated antibodies.E-H LDH release(E), IL-1β secretion(F), IL-18 secretion (G) and TNF-ɑ secretion (H) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; **, P < 0.01; ***, P <0.001; In each panel, data are representative of at least three independent experiments.


Figure 1B . Relative quantification of all four histone proteins in each tissue replicate ( order of appearance per replicate : H2A , H2B , H3 . 3 and H4 ) . C . Immunohistochemistry images from the Human Protein Atlas ( www . proteinatlas . org ) for protein ANXA1 with nuclear staining ( blue ) for three selected tissues . D . Calibration curves for two of the four histone peptides , with decreasing amount of QPrEST standard spiked into a U2OS cell lysate .

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Figure 1B. Relative quantification of all four histone proteins in each tissue replicate (order of appearance per replicate: H2A, H2B, H3.3 and H4).C. Immunohistochemistry images from the Human Protein Atlas (www.proteinatlas.org) for protein ANXA1 with nuclear staining (blue) for three selected tissues.D. Calibration curves for two of the four histone peptides, with decreasing amount of QPrEST standard spiked into a U2OS cell lysate.


Figure 2A . Absolute copy number of protein in kidney tissue and human embryonal kidney cells ( HEK293 ) , liver tissue and liver cancer cell line ( HepG2 ) , lung tissue and lung cancer cell line ( A549 ) , and breast tissue and breast cancer cell line ( MCF7 ) . The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue . B . The direct correlation between RNA ( TPM ) and protein abundances ( copy number ) for all quantified genes in the same tissues and cell lines . Spearman ( ρ ) and Pearson ( r ) correlation between the two values across the quantified genes are shown . The other seven tissues and five cell lines are shown in Figure EV4 .

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Figure 2A. Absolute copy number of protein in kidney tissue and human embryonal kidney cells (HEK293), liver tissue and liver cancer cell line (HepG2), lung tissue and lung cancer cell line (A549), and breast tissue and breast cancer cell line (MCF7). The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue.B. The direct correlation between RNA (TPM) and protein abundances (copy number) for all quantified genes in the same tissues and cell lines. Spearman (ρ) and Pearson (r) correlation between the two values across the quantified genes are shown. The other seven tissues and five cell lines are shown in Figure EV4.


Figure 3Subcellular localization by immunofluorescence staining and immunohistochemistry staining in tissue sections by three different antibodies ( SELENBP1 , HPA011731 ; STOM , HPA010961 ; ASS1 , HPA020896 ) . Microtubule - and nuclear probes are visualized in red and blue respectively . Antibody staining is shown in green . RNA to protein ratio across nine cell lines and 11 tissues with Spearman rho , Pearson r and R2 for each gene . All other genes can be found in Figure EV1 .

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Figure 3Subcellular localization by immunofluorescence staining and immunohistochemistry staining in tissue sections by three different antibodies (SELENBP1, HPA011731; STOM, HPA010961; ASS1, HPA020896). Microtubule- and nuclear probes are visualized in red and blue respectively. Antibody staining is shown in green. RNA to protein ratio across nine cell lines and 11 tissues with Spearman rho, Pearson r and R2 for each gene. All other genes can be found in Figure EV1.


Figure 1A : Volcano plot showing significantly regulated genes between control and ATP - treated msEGCs . G - I : qPCR analysis of indicated gliosis genes in ATP - treated EGCs . J : qPCR analysis of IL - 6 in msEGCs that were treated for 6h with ATP . K : IL - 6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP .

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Figure 1A: Volcano plot showing significantly regulated genes between control and ATP-treated msEGCs.G-I: qPCR analysis of indicated gliosis genes in ATP-treated EGCs.J: qPCR analysis of IL-6 in msEGCs that were treated for 6h with ATP.K: IL-6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP.


Figure 2A : Effect of P2 receptor antagonism on ATP induced IL - 6 release . Cells were treated with P2 - antagonist suramin ( 1 , 10 and 100µM ) alone or together with ATP ( 100µM ) for 24h . B : ATP induced IL - 6 release in msEGCs measured by ELISA . Cells were treated with the indicated concentrations of ATP - and ATPɣS for 24h . C : Effects of P2X antagonists on ATP induced IL - 6 release . Cells were treated for 24h alone or together with ATP ( 100µM ) in absence or presence of P2X2 , P2X4 and P2X7 - antagonists PSB - 1011 , 5 - BD - BD and A740003 , respectively . D : P2X2 antagonism of ATP induced mRNA expression of IL - 6 , GFAP and RCAN by qPCR in msEGCs . Cells were treated with the P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 10µM ) for 6h . E : Representative confocal images of P2X2 ( green ) and GFAP ( violet ) positive msEGCs in vivo and in vitro . White arrows mark double positive ( white ) cells . Scale bar 50µm . F : P2X2 - siRNA reduces P2X2 - mRNA and dampens the gliosis gene expression after ATPɣS ( 100µM ) treatment for 6h . G : P2X2 - siRNA reduces IL - 6 release after ATPɣS treatment ( 10µM , 100µM ) for 6h .

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Figure 2A: Effect of P2 receptor antagonism on ATP induced IL-6 release. Cells were treated with P2-antagonist suramin (1, 10 and 100µM) alone or together with ATP (100µM) for 24h.B: ATP induced IL-6 release in msEGCs measured by ELISA. Cells were treated with the indicated concentrations of ATP-and ATPɣS for 24h.C: Effects of P2X antagonists on ATP induced IL-6 release. Cells were treated for 24h alone or together with ATP (100µM) in absence or presence of P2X2, P2X4 and P2X7-antagonists PSB-1011, 5-BD-BD and A740003, respectively.D: P2X2 antagonism of ATP induced mRNA expression of IL-6, GFAP and RCAN by qPCR in msEGCs. Cells were treated with the P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (10µM) for 6h.E: Representative confocal images of P2X2 (green) and GFAP (violet) positive msEGCs in vivo and in vitro. White arrows mark double positive (white) cells. Scale bar 50µm.F: P2X2-siRNA reduces P2X2-mRNA and dampens the gliosis gene expression after ATPɣS (100µM) treatment for 6h.G: P2X2-siRNA reduces IL-6 release after ATPɣS treatment (10µM, 100µM) for 6h.


Figure 3A : ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation ( IM ) or were naïve . B : PCA plot of gene expression from POI mice at different disease stages and naïve mice ; n = 3 for each group . F - H : qPCR analysis of indicated gliosis marker in mice that underwent IM ( n = 7 ) . I : Histological analysis of EGC proliferation in vivo . Representative confocal images and quantification show Sox10 ( green ) and Ki67 ( violet ) positive EGCs ( white arrows ) in the small bowel muscularis externa at naïve , IM6h and 24h . Scale bar 50µm . K - M : qPCR analysis of indicated gliosis markers in td + glia cells from naïve mice and mice that underwent IM ( n = 5 - 7 , POI mice ) .

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Figure 3A: ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation (IM) or were naïve.B: PCA plot of gene expression from POI mice at different disease stages and naïve mice; n=3 for each group.F-H: qPCR analysis of indicated gliosis marker in mice that underwent IM (n=7).I: Histological analysis of EGC proliferation in vivo. Representative confocal images and quantification show Sox10 (green) and Ki67 (violet) positive EGCs (white arrows) in the small bowel muscularis externa at naïve, IM6h and 24h. Scale bar 50µm.K-M: qPCR analysis of indicated gliosis markers in td+ glia cells from naïve mice and mice that underwent IM (n=5-7, POI mice).


Figure 4Postoperative gene expression analyses of indicated gliosis marker in the ME . Postoperative gene expression analyses of indicated gliosis marker in the ME . E : Histological analysis of EGC proliferation by quantification of Ki67 ( violet ) and Sox10 positive EGCs at IM24h . Mice were treated as seen in ( A ) . F : Histological counting of infiltrating myeloperoxidase ( MPO ) - positive leukocytes in the postoperative ME ( n = 6 - 8 ) . G : FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle . CD45 , Ly6C and CX3CR1 were used to distinguish resident macrophages ( CD45 + / Ly6C - / CX3CR1 + ) , infiltrating monocytes ( CD45 + / Ly6C + / CX3CR1 - ) and infiltrated monocyte - derived macrophages ( CD45 + / Ly6C + / CX3CR1 + ) . H : Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle .

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Figure 4Postoperative gene expression analyses of indicated gliosis marker in the ME.Postoperative gene expression analyses of indicated gliosis marker in the ME.E: Histological analysis of EGC proliferation by quantification of Ki67 (violet) and Sox10 positive EGCs at IM24h. Mice were treated as seen in (A).F: Histological counting of infiltrating myeloperoxidase (MPO)-positive leukocytes in the postoperative ME (n=6-8).G: FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle. CD45, Ly6C and CX3CR1 were used to distinguish resident macrophages (CD45+/Ly6C-/CX3CR1+), infiltrating monocytes (CD45+/Ly6C+/CX3CR1-) and infiltrated monocyte-derived macrophages (CD45+/Ly6C+/CX3CR1+).H: Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle.


Figure 5E : IL - 6 protein release in hEGC cultures upon stimulation with ATP ( 200µM ) or ATPɣS ( 100µM ) after 6h and 24h treatment ( n = 4 - 6 , hEGCs ) . F : IL - 6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations ; n = 4 - 6 , hEGCs . G : Immunofluorescence microscopy revealed P2X2 expression ( green ) in a majority of s100β + ( violet ) hEGCs in intact myenteric ganglia of the human colon . White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons . Scale bar , 50µm . H : Effect of the P2X2 antagonism on ATP - induced IL - 6 release . ELISA measurement of IL - 6 in hEGCs upon treatment with P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 200µM ) treated for 24h ( n = 6 , hEGCs ) . I : PSB - 1011 ( 20μM ) treatment inhibited ATP - triggered calcium responses in HEK cells transfected with P2X2 . Data are represented as ΔF / F0 + SEM ; n = 102 HEK cells . J : The P2X2 receptor antagonist PSB - 0711 ( 20μM ) nearly abolished the ATP - triggered calcium response in HEK cells transfected with P2X2 . n = 219 HEK cells . K : Ambroxol blocks ATP - induced IL - 6 release in hEGCs ; Protein release measurement by ELISA of IL - 6 in hEGCs . Cells were treated with ATP ( 200 µM ) alone or together with ambroxol ( 20 µM ) for 24h ; n = 6 , hEGCs . L - N : qPCR analysis of several gliosis and ATP - target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol ( 20µM ) . ( n = 7 , 4 human patients ) .

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Figure 5E: IL-6 protein release in hEGC cultures upon stimulation with ATP (200µM) or ATPɣS (100µM) after 6h and 24h treatment (n=4-6, hEGCs).F: IL-6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations; n=4-6, hEGCs.G: Immunofluorescence microscopy revealed P2X2 expression (green) in a majority of s100β+ (violet) hEGCs in intact myenteric ganglia of the human colon. White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons. Scale bar, 50µm.H: Effect of the P2X2 antagonism on ATP-induced IL-6 release. ELISA measurement of IL-6 in hEGCs upon treatment with P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (200µM) treated for 24h (n=6, hEGCs).I: PSB-1011 (20μM) treatment inhibited ATP-triggered calcium responses in HEK cells transfected with P2X2. Data are represented as ΔF/F0 +SEM; n=102 HEK cells. J: The P2X2 receptor antagonist PSB-0711 (20μM) nearly abolished the ATP-triggered calcium response in HEK cells transfected with P2X2. n=219 HEK cells.K: Ambroxol blocks ATP-induced IL-6 release in hEGCs; Protein release measurement by ELISA of IL-6 in hEGCs. Cells were treated with ATP (200 µM) alone or together with ambroxol (20 µM) for 24h; n=6, hEGCs.L-N: qPCR analysis of several gliosis and ATP-target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol (20µM). (n=7, 4 human patients).


Figure 1 ( C ) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA . The dCas9 - ω was verified in the ∆ rpoZ strain background , and the rest of candidates were verified in the wild - type strain ( BW25113 ) . ( D ) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9 - AsiA with gRNA - H3 under kanamycin selection ( 2 . 5 μg / ml ) . ( E ) Different gRNAs were paired with dCas9 - AsiA and dCas9 - ω to profile the optimal gRNA binding distance . The dCas9 - AsiA was verified in the wild - type strain ( BW25113 ) , and dCas9 - ω was examined in the ∆ rpoZ strain background . ( F ) Comparing single and multiple gRNA with dCas9 - AsiA .

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Figure 1(C) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA. The dCas9-ω was verified in the ∆rpoZ strain background, and the rest of candidates were verified in the wild-type strain (BW25113).(D) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9-AsiA with gRNA-H3 under kanamycin selection (2.5 μg/ml). (E) Different gRNAs were paired with dCas9-AsiA and dCas9-ω to profile the optimal gRNA binding distance. The dCas9-AsiA was verified in the wild-type strain (BW25113), and dCas9-ω was examined in the ∆rpoZ strain background. (F) Comparing single and multiple gRNA with dCas9-AsiA.


Figure 2 ( C ) Distribution of fluorescence signal of the GFP reporter induced by different dCas9 - AsiA variants ( top ) . Fold induction by different dCas9 - AsiA variants is shown ( bottom ) . ( D ) CRISPRa induction of promoters with varying basal expression levels . CRISPRa - , basal expression of the promoter ; CRISPRa + , expression activated by dCas9 - AsiA - m2 . 1 and associated gRNA - H4 .

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Figure 2 (C) Distribution of fluorescence signal of the GFP reporter induced by different dCas9-AsiA variants (top). Fold induction by different dCas9-AsiA variants is shown (bottom).  (D) CRISPRa induction of promoters with varying basal expression levels. CRISPRa-, basal expression of the promoter; CRISPRa+, expression activated by dCas9-AsiA-m2.1 and associated gRNA-H4.


Figure 3 ( A ) CasTA2 . 1 upregulates a genomically inserted GFP reporter . ( B ) Chromosomal gene targets activated by CasTA2 . 1 with bars showing the activation fold change and dots showing basal expression of each gene . Expression was quantified using RT - qPCR . ( C ) CasTA2 . 1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene . A non - specific gRNA is used as the negative control . ( D ) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2 . 1 on a reporter containing GFP and mScarlet . Parental cells had low basal GFP and high basal mScarlet expression .

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Figure 3(A) CasTA2.1 upregulates a genomically inserted GFP reporter. (B) Chromosomal gene targets activated by CasTA2.1 with bars showing the activation fold change and dots showing basal expression of each gene. Expression was quantified using RT-qPCR. (C) CasTA2.1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene. A non-specific gRNA is used as the negative control. (D) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2.1 on a reporter containing GFP and mScarlet. Parental cells had low basal GFP and high basal mScarlet expression.


Figure 4 ( B ) Promoters significantly activated by CasTA2 . 1 using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted with basal expression level on x - axis against fold activation by CRISPRa on y - axis . N is the total number of promoters shown . Red box corresponds to highly activated promoters ( fold change > 10 ) . ( C ) Highly activated promoters ( > 10 - fold ) using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted . Basal expression levels ( purple lines ) , induced expression level ( orange lines on left or red lines on right , activated with gRNA - H23 or gRNA - H22 , respectively ) and induced fold changes ( gray bars ) are shown . Promoters were first ranked and grouped by basal expression levels . Within each group , promoters were ranked by activation fold changes .

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Figure 4 (B) Promoters significantly activated by CasTA2.1 using gRNA-H23 (left) or gRNA-H22 (right) are plotted with basal expression level on x-axis against fold activation by CRISPRa on y-axis. N is the total number of promoters shown. Red box corresponds to highly activated promoters (fold change >10).   (C) Highly activated promoters (>10-fold) using gRNA-H23 (left) or gRNA-H22 (right) are plotted. Basal expression levels (purple lines), induced expression level (orange lines on left or red lines on right, activated with gRNA-H23 or gRNA-H22, respectively) and induced fold changes (gray bars) are shown. Promoters were first ranked and grouped by basal expression levels. Within each group, promoters were ranked by activation fold changes.