Figure 3 ( A ) Hematoxylin and eosin ( H & E , upper panels ) and periodic acid Schiff ( PAS , lower panels ) staining of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bars : 500 µm . ( B ) Computational analysis of PAS staining ( n ≥ 4 mice / group ) . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M . ( C ) Quantification of hepatic glycogen in vehicle ( n = 6 ) - and TB - 1 - treated AslNeo / Neo mice ( n = 12 ) compared to WT ( n = 5 ) controls . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M . ( D ) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo / Neo mice treated with either with TB - 1 or vehicle ( n = 4 mice / group ) . * p < 0 . 05 ( One - way ANOVA ) . GAPDH was used as loading control . Data information : All values are shown as averages ± S . E . M . ( E ) Representative electron microscopy images of liver samples harvested from WT and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bar : 900 nm .

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 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 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 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

Figure 3(A) Hematoxylin and eosin (H&E, upper panels) and periodic acid Schiff (PAS, lower panels) staining of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bars: 500 µm.(B) Computational analysis of PAS staining (n≥4 mice/group). **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.(C) Quantification of hepatic glycogen in vehicle (n=6)- and TB-1-treated AslNeo/Neo mice (n=12) compared to WT (n=5) controls. **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.(D) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo/Neo mice treated with either with TB-1 or vehicle (n=4 mice/group). *p<0.05 (One-way ANOVA). GAPDH was used as loading control. Data information: All values are shown as averages ± S.E.M.(E) Representative electron microscopy images of liver samples harvested from WT and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bar: 900 nm.


Figure 4 ( A ) Representative electron microscopy images of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . False color on the images indicates glycogen within the nuclei . Scale bar : 1 μm . ( B ) Quantification of nuclei containing glycogen ( approx . 180 nuclei were analyzed in total per condition , n = 3 mice / group ) .

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

Figure 4(A) Representative electron microscopy images of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. False color on the images indicates glycogen within the nuclei. Scale bar: 1 μm. (B) Quantification of nuclei containing glycogen (approx. 180 nuclei were analyzed in total per condition, n=3 mice/group).


Figure 1 ( A ) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h . Then , WB was used to measure p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein expression . " Nor " = normal medium . Actin served as the loading control . The red text " - Cys " indicates that cystine deficiency most significantly activates AMPK and its substrates . ( F ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein in 293T cells transfected with shRNAs targeting LKB1 , CaMKK2 , TAK1 or a non - targeting control ( NTC ) that were treated with cystine - deficient medium for 8 h . Actin served as the loading control . ( I ) 293T cells cultured with cystine - deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti - AMPKα , followed by WB analysis with anti - AMPKα and anti - CaMKK2 .

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

Figure 1 (A) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h. Then, WB was used to measure p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein expression. "Nor" = normal medium. Actin served as the loading control. The red text "-Cys" indicates that cystine deficiency most significantly activates AMPK and its substrates. (F) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells transfected with shRNAs targeting LKB1, CaMKK2, TAK1 or a non-targeting control (NTC) that were treated with cystine-deficient medium for 8 h. Actin served as the loading control. (I) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-AMPKα, followed by WB analysis with anti-AMPKα and anti-CaMKK2.


Figure 2 ( E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK ACC , Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine - deficient medium for 8 h . Actin served as the loading control . ( J ) WB analysis of p - AMPK and total AMPK protein expression in CARS - overexpressing 293T cells and RCC4 cells that were treated with 1 µg / ml STO - 609 or DMSO for 8 h . Actin served as the loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK ACC, Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine-deficient medium for 8 h. Actin served as the loading control.  (J) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells and RCC4 cells that were treated with 1 µg/ml STO-609 or DMSO for 8 h. Actin served as the loading control.


Figure 3 ( D , E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor , AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine - deficient medium for 8 h ( D ) or transfected with Flag - CARS for 48 h ( E ) . Actin served as the loading control . ( F ) 293T cells were transfected with HA - AMPKγ2 alone or with Flag - CARS for 48 h and then cultured with cystine - deficient medium or complete medium for 8 h . Cell lysates were immunoprecipitated with anti - Flag , and a WB analysis was performed .

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

Figure 3 (D, E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine-deficient medium for 8 h (D) or transfected with Flag-CARS for 48 h (E). Actin served as the loading control. (F) 293T cells were transfected with HA-AMPKγ2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed.


Figure 4 ( B ) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups . ( C , D ) The crystal violet assay ( C ) and apoptosis rate assay ( D ) were performed in 293T cells transfected with shRNAs targeting NTC , CARS , CaMKK2 or AMPKγ2 and further treated with cystine - deficient medium for 24 h with or without 1 mM AICAR . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups . ( F WB analysis of p - AMPK , total AMPK , CARS , AMPKγ2 , and CaMKK2 protein expression with 786 - O cells transfected with Flag - EV , Flag - CARS , Flag - CaMKK2 or Flag - AMPKγ2 and further treated with cystine - deficient medium for 8 h . Actin served as the loading control . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(B) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.(C, D) The crystal violet assay (C) and apoptosis rate assay (D) were performed in 293T cells transfected with shRNAs targeting NTC, CARS, CaMKK2 or AMPKγ2 and further treated with cystine-deficient medium for 24 h with or without 1 mM AICAR. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups. (F WB analysis of p-AMPK, total AMPK, CARS, AMPKγ2, and CaMKK2 protein expression with 786-O cells transfected with Flag-EV, Flag-CARS, Flag-CaMKK2 or Flag-AMPKγ2 and further treated with cystine-deficient medium for 8 h. Actin served as the loading control. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


Figure 1 ( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) produced with lopinavir ( LPV , 0 - 100 nM ) detecting p24 antibody . Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated THP - 1 shSAMHD1 cells . Mean ± SD , n = 3 . Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated U87 cells . Mean ± SD , n = 3 . ISG qPCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . ISG qPCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . ISG qPCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . G ) CXCL - 10 protein in cell supernatants from D - F ( ELISA ) . ( H ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 2 U RT / ml 30 nM LPV - treated HIV - 1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib . A control was stimulated with 1 ng / ml IFNβ . ( I ) RT products from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 3 U RT / ml LPV - treated HIV - 1 GFP viruses . ( J ) Immunoblot of HIV - 1 R9 BaL virus particles ( 2 × 1011 genomes ) produced with LPV ( 0 - 100 nM ) detecting p24 . ( K , L ) ISG qRT - PCR ( K ) and infection data ( L ) from primary monocyte - derived macrophages ( MDM ) infected for 24 h with LPV - treated HIV - 1 R9 BaL viruses ( 0 . 2 U RT / ml ) . Data are collated from two donors ( represented by circles and squares ) , n = 3 . Horizontal line represents the median . For experiments in which the virus dose used was normalised by RT activity , the number of genome copies was also measured by qPCR of virus . This gave dose equivalents of within 2 - 3 fold of RT equivalents .

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 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 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 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 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 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 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 1(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) produced with lopinavir (LPV, 0-100 nM) detecting p24 antibody.Titration of LPV-treated HIV-1 GFP viruses on PMA-treated THP-1 shSAMHD1 cells. Mean ± SD, n=3.Titration of LPV-treated HIV-1 GFP viruses on PMA-treated U87 cells. Mean ± SD, n=3. ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).  ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).  ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). G) CXCL-10 protein in cell supernatants from D-F (ELISA).(H) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ.(I) RT products from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.3 U RT/ml LPV-treated HIV-1 GFP viruses.(J) Immunoblot of HIV-1 R9 BaL virus particles (2×1011 genomes) produced with LPV (0-100 nM) detecting p24.(K, L) ISG qRT-PCR (K) and infection data (L) from primary monocyte-derived macrophages (MDM) infected for 24 h with LPV-treated HIV-1 R9 BaL viruses (0.2 U RT/ml). Data are collated from two donors (represented by circles and squares), n=3. Horizontal line represents the median. For experiments in which the virus dose used was normalised by RT activity, the number of genome copies was also measured by qPCR of virus. This gave dose equivalents of within 2-3 fold of RT equivalents.


Figure 2 ( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) with varying proportions of ΔCA - SP1 protease cleavage mutation detecting p24 . ( B ) Titration of HIV - 1 GFP ΔCA - SP1 viruses on U87 cells . Mean ± SD , n = 3 . ( C ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . D ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . E ) CXCL - 10 protein in supernatants from ( C , D ) ( ELISA ) . ( F ) RT products from THP - 1 cells transduced for 24 h with 6 × 109 genomes / ml ( approx . 0 . 5 U RT / ml ) HIV - 1 GFP ΔCA - SP1 viruses . ( G ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 016 - 0 . 2 U RT / ml ) . Data are shown as individual measurements , representative of 2 repeats . ( H ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 mutant , or stimulated with 4 μg / ml cGAMP as a control , in the presence of DMSO vehicle or 2 μM ruxolitinib . ( I ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 3 × 108 genomes / ml or 1 . 5 × 109 genomes / ml , equivalent to 0 . 1 U RT / ml and 0 . 5 U RT / ml ) . ( J ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib . primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib . K ) CXCL - 10 protein in supernatants from ( J ) ( ELISA ) .

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 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 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 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 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 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 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 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 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 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

Figure 2(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) with varying proportions of ΔCA-SP1 protease cleavage mutation detecting p24.(B) Titration of HIV-1 GFP ΔCA-SP1 viruses on U87 cells. Mean ± SD, n=3.(C ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).D) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). E) CXCL-10 protein in supernatants from (C, D) (ELISA).(F) RT products from THP-1 cells transduced for 24 h with 6×109 genomes/ml (approx. 0.5 U RT/ml) HIV-1 GFP ΔCA-SP1 viruses.(G) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.016 - 0.2 U RT/ml). Data are shown as individual measurements, representative of 2 repeats.(H) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 mutant, or stimulated with 4 μg/ml cGAMP as a control, in the presence of DMSO vehicle or 2 μM ruxolitinib.(I) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (3×108 genomes/ml or 1.5×109 genomes/ml, equivalent to 0.1 U RT/ml and 0.5 U RT/ml).(J) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib.primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib. K) CXCL-10 protein in supernatants from (J) (ELISA).


Figure 3 ( A ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . B ) CXCL - 10 protein in supernatants from ( A ) ( ELISA ) . ISG qRT - PCR from THP - 1 - IFIT - 1 cells transduced for 24 h with 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 109 and 3 × 109 genomes / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . ISG qRT - PCR from THP - 1 - IFIT - 1 cells transduced for 24 h with 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 109 and 3 × 109 genomes / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . ( E ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ΔCA - SP1 ( WT ) , 75 % ΔCA - SP1 , 75 % ΔCA - SP1 carrying a mutation in reverse transcriptase ( 75 % ΔCA - SP1 RT D185E ) or 75 % ΔCA - SP1 carrying a mutation in integrase ( 75 % ΔCA - SP1 INT D116N ) ( 3 . 75 × 109 , 7 . 5 × 109 and 1 . 5 × 1010 genomes / ml ) . ( F ) CXCL - 10 protein in supernatants from ( E ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

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 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 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 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

Figure 3(A) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir. B) CXCL-10 protein in supernatants from (A) (ELISA).ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.(E) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % ΔCA-SP1 (WT), 75 % ΔCA-SP1, 75 % ΔCA-SP1 carrying a mutation in reverse transcriptase (75 % ΔCA-SP1 RT D185E) or 75 % ΔCA-SP1 carrying a mutation in integrase (75 % ΔCA-SP1 INT D116N) (3.75×109, 7.5×109 and 1.5×1010 genomes/ml).(F) CXCL-10 protein in supernatants from (E) (ELISA). Triangles indicate CXCL-10 not detected.


Figure 4 ( A ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING or MAVS , or a gRNA control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP 75 % ΔCA - SP1 ( 0 . 4 U RT / ml ) or stimulated by transfection with either HT - DNA ( 0 . 1 μg / ml ) or poly I : C ( 0 . 5 μg / ml ) . IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line stimulated for 24 h with poly I : C ( transfection , 0 . 5 μg / ml ) , HT - DNA ( transfection , 0 . 1 μg / ml ) , LPS ( 50 ng / ml ) or cGAMP ( transfection , 0 . 5 μg / ml ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml ) PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml ) ( C ) . D ) CXCL - 10 protein in supernatants from ( C ) ( ELISA ) . Triangles indicate CXCL - 10 not detected . ( E ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . ( F ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . G ) CXCL - 10 protein in supernatants from ( E ) . PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . ( H ) CXCL - 10 protein in supernatants from ( F ) ( ELISA ) . ( I ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 control cells transduced for 48 h with WT , DRV - treated ( DRV , 12 . 5 nM ) or HIV - 1 GFP containing 90 % ΔCA - SP1 ( 1 × 1010 genomes / ml ) in the presence of DMSO vehicle , 2 μM ruxolitinib or 0 . 5 μg / ml H151 .

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 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 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 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 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 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 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

Figure 4(A) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING or MAVS, or a gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP 75 % ΔCA-SP1 (0.4 U RT/ml) or stimulated by transfection with either HT-DNA (0.1 μg/ml) or poly I:C (0.5 μg/ml).IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line stimulated for 24 h with poly I:C (transfection, 0.5 μg/ml), HT-DNA (transfection, 0.1 μg/ml), LPS (50 ng/ml) or cGAMP (transfection, 0.5 μg/ml)IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml)PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml) (C). D) CXCL-10 protein in supernatants from (C) (ELISA). Triangles indicate CXCL-10 not detected.(E) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).(F) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). G) CXCL-10 protein in supernatants from (E).PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). (H) CXCL-10 protein in supernatants from (F) (ELISA).(I) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 control cells transduced for 48 h with WT, DRV-treated (DRV, 12.5 nM) or HIV-1 GFP containing 90 % ΔCA-SP1 (1×1010 genomes/ml) in the presence of DMSO vehicle, 2 μM ruxolitinib or 0.5 μg/ml H151.


Figure 5Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with a fixed dose of HIV - 1 GFP ( 5 × 107 genomes / ml ) and increasing doses of HIV - LUC ΔCA - SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with LPV - treated HIV - LUC viruses Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 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

Figure 5Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with a fixed dose of HIV-1 GFP (5×107 genomes/ml) and increasing doses of HIV-LUC ΔCA-SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with LPV-treated HIV-LUC viruses Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.


Figure 6 ( A ) Infection data for THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . B ) IFIT - 1 reporter activity in supernatant from ( A ) . ISG qRT - PCR from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 25 - 1 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . ISG qRT - PCR from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 25 - 1 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . ( E ) CXCL - 10 protein in supernatants of THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 3 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . ( F ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) and ruxolitinib ( Rux , 2 μM ) as indicated . ( G ) IRF reporter activity from THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP ( 3 U / ml and 6 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . ( H ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells lacking MAVS or a matching gRNA control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP ( 3 U / ml and 6 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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 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 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 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

Figure 6(A) Infection data for THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM). B) IFIT-1 reporter activity in supernatant from (A).ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).(E) CXCL-10 protein in supernatants of THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).(F) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM) and ruxolitinib (Rux, 2 μM) as indicated.(G) IRF reporter activity from THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).(H) IFIT-1 reporter activity from THP-1-IFIT-1 cells lacking MAVS or a matching gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).


Figure 1Depletion of RBM14 induces ectopic formation of centriolar protein complexesA - E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) and C - Nap1 ( magenta ) ( A ) , Centrobin ( green ) and C - Nap1 ( magenta ) ( B ) , centrin - 2 ( green ) and CPAP ( magenta ) ( C ) , acetylated - tubulin ( green ) and CP110 ( magenta ) ( D ) , and centrin - 2 ( green ) and γ - tubulin ( magenta ) ( E ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition . Values are mean percentages ± standard error of mean ( SEM ) from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 , * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) . Note that we counted the number of C - Nap1 foci after anaphase in mitosis when C - Nap1 signals recover from the reduction in prometaphase .

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

Figure 1Depletion of RBM14 induces ectopic formation of centriolar protein complexesA-E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta) (A), Centrobin (green) and C-Nap1 (magenta) (B), centrin-2 (green) and CPAP (magenta) (C), acetylated-tubulin (green) and CP110 (magenta) (D), and centrin-2 (green) and γ-tubulin (magenta) (E). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01, n.s., not significant (one-tailed t-test). Note that we counted the number of C-Nap1 foci after anaphase in mitosis when C-Nap1 signals recover from the reduction in prometaphase.


Figure 2A U2OS cells or U2OS cells expressing FLAG - RBM14 full length ( FL , aa1 - 669 ) , N - terminal fragment ( RBM14 [ N ] , aa1 - 150 ) or C - terminal fragment ( RBM14 [ C ] , aa151 - 669 ) and treated with control siRNA or siRNA against 3 ′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin - 2 . Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition . Values are mean percentages ± SEM from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) . C , D Cytoplasmic RBM14 could suppress centriole amplification in HU - treated cells . ( C ) U2OS cells or U2OS cells expressing FLAG - RBM14 FL or [ C ] and treated with or without HU were stained with antibodies against centrin - 2 ( green ) and FLAG ( magenta ) . ( D ) U2OS cells or U2OS cells expressing FLAG - RBM14 FL , FLAG - RBM14 - NES or GFP - RBM14 - PACT and treated with or without HU were stained with antibodies against FLAG ( magenta , left ) or GFP ( magenta , right ) as well as centrin - 2 ( green ) . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 10 μm . Histograms represent frequency of cells in interphase with excess centrin foci in each condition . The percentages of U2OS cells with centrosomal localization of the RBM14 full - length protein or mutants are shown below the immunofluorescence images . We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . Please note that cytoplasmic expression levels of GFP - RBM14 - PACT are less than those of FLAG - RBM14 FL ( ˜ 0 . 5 - fold ) or FLAG - RBM14 - NES ( ˜ 0 . 7 - fold ) .

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 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

Figure 2A U2OS cells or U2OS cells expressing FLAG-RBM14 full length (FL, aa1-669), N-terminal fragment (RBM14[N], aa1-150) or C-terminal fragment (RBM14[C], aa151-669) and treated with control siRNA or siRNA against 3′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin-2. Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).C, D Cytoplasmic RBM14 could suppress centriole amplification in HU-treated cells. (C) U2OS cells or U2OS cells expressing FLAG-RBM14 FL or [C] and treated with or without HU were stained with antibodies against centrin-2 (green) and FLAG (magenta). (D) U2OS cells or U2OS cells expressing FLAG-RBM14 FL, FLAG-RBM14-NES or GFP-RBM14-PACT and treated with or without HU were stained with antibodies against FLAG (magenta, left) or GFP (magenta, right) as well as centrin-2 (green). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 10 μm. Histograms represent frequency of cells in interphase with excess centrin foci in each condition. The percentages of U2OS cells with centrosomal localization of the RBM14 full-length protein or mutants are shown below the immunofluorescence images. We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Please note that cytoplasmic expression levels of GFP-RBM14-PACT are less than those of FLAG-RBM14 FL (˜0.5-fold) or FLAG-RBM14-NES (˜0.7-fold).


Figure 3A HeLa cells immunoprecipitated with control IgG or STIL antibodies . Soluble cytosolic fractions ( input ) and immunoprecipitates ( IPs ) were analyzed by Western blotting using RBM14 , STIL or CPAP antibodies . B GST pull - down assay testing interactions between purified STIL [ N ] ( ˜ 5 μg , aa1 - 1018 ) and GST - RBM14 [ N ] or [ C ] . The asterisks indicate non - specific bands . D Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) and STIL ( magenta ) . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess STIL foci co - localized with centrin foci . Values are mean percentages ± SEM from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . E In vitro competitive binding assay . GST pull - down experiment was performed as in ( B ) , with purified STIL [ N ] and GST - RBM14 [ C ] in the presence of the indicated amount of purified His - CPAP [ SBD ] , His - tagged STIL - Binding Domain of CPAP . The fraction of STIL [ N ] bound to GST - RBM14 [ C ] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N - terminal region of STIL . Input materials were analyzed by Western blotting using the STIL or CPAP antibodies . The precipitated GST - RBM14 [ C ] was analyzed by SDS - PAGE , stained with SimplyBlue ™ Safe ( Invitrogen ) . F Interaction between STIL and GFP - CPAP in the presence of FLAG - RBM14 FL or fragments . U2OS cells expressing GFP - CPAP were transfected with empty vector as a control , FLAG - RBM14 [ N ] , FLAG - RBM14 [ C ] or FLAG - RBM14 FL constructs , immunoprecipitated with STIL antibodies , and the resulting IPs were analyzed by Western blotting using STIL , CPAP or FLAG antibodies . Quantification of relative protein amounts of co - immunoprecipitated GFP - CPAP in each STIL - IP fraction , normalized with the amount of STIL precipitated , is shown below the panels . Means ± SEM were calculated from three independent experiments . * * P < 0 . 01 ( one - tailed t - test ) . Note that we did not find any significant effect of exogenous expression of RBM14 full - length protein or deletion mutants on the expression levels of GFP - CPAP or endogenous STIL proteins in this experiment . G U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin - 2 ( green ) and C - Nap1 ( magenta ) . Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . Representative mitotic cells treated with siRNAs against RBM14 alone , RBM14 + STIL or RBM14 + CPAP are shown below . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm .

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 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 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 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 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 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

Figure 3A HeLa cells immunoprecipitated with control IgG or STIL antibodies. Soluble cytosolic fractions (input) and immunoprecipitates (IPs) were analyzed by Western blotting using RBM14, STIL or CPAP antibodies.B GST pull-down assay testing interactions between purified STIL[N] (˜5 μg, aa1-1018) and GST-RBM14 [N] or [C]. The asterisks indicate non-specific bands.D Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin-2 (green) and STIL (magenta). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess STIL foci co-localized with centrin foci. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01 (one-tailed t-test).E In vitro competitive binding assay. GST pull-down experiment was performed as in (B), with purified STIL[N] and GST-RBM14[C] in the presence of the indicated amount of purified His-CPAP[SBD], His-tagged STIL-Binding Domain of CPAP. The fraction of STIL[N] bound to GST-RBM14[C] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N-terminal region of STIL. Input materials were analyzed by Western blotting using the STIL or CPAP antibodies. The precipitated GST-RBM14[C] was analyzed by SDS-PAGE, stained with SimplyBlue™ Safe (Invitrogen).F Interaction between STIL and GFP-CPAP in the presence of FLAG-RBM14 FL or fragments. U2OS cells expressing GFP-CPAP were transfected with empty vector as a control, FLAG-RBM14[N], FLAG-RBM14[C] or FLAG-RBM14 FL constructs, immunoprecipitated with STIL antibodies, and the resulting IPs were analyzed by Western blotting using STIL, CPAP or FLAG antibodies. Quantification of relative protein amounts of co-immunoprecipitated GFP-CPAP in each STIL-IP fraction, normalized with the amount of STIL precipitated, is shown below the panels. Means ± SEM were calculated from three independent experiments. **P < 0.01 (one-tailed t-test). Note that we did not find any significant effect of exogenous expression of RBM14 full-length protein or deletion mutants on the expression levels of GFP-CPAP or endogenous STIL proteins in this experiment.G U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta). Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Representative mitotic cells treated with siRNAs against RBM14 alone, RBM14 + STIL or RBM14 + CPAP are shown below. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm.


Figure 4A U2OS cells treated with control siRNA , RBM14 siRNA alone or RBM14 + HsSAS - 6 siRNAs were stained with antibodies against centrin - 2 ( green ) and HsSAS - 6 ( magenta ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . n . s . , not significant ( one - tailed t - test ) . B Live imaging of cycling RBM14 - depleted HeLa cells expressing GFP - centrin1 ( green ) and RFP - H2B ( magenta ) . Insets show approximately 1 . 5 - fold magnified images of fluorescent foci . Time is denoted in hh : min , and scale bar is 10 μm . Time zero corresponds to the onset of excess centrin foci formation . Quantification of the number and total signal intensity of centrin foci in HeLa cells treated with control or RBM14 siRNA over time . Means ± SEM are shown ( n = 5 ) . C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase . Time - lapse recording across the cell cycle of HeLa cells expressing GFP - centrin ( green ) and RFP - H2B ( magenta ) and treated with RBM14 siRNA . Time is denoted in hh : min , and scale bar is 10 μm . Time zero corresponds to the metaphase onset . D Ectopic formation of centriolar protein complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment . Insets show threefold magnified images of fluorescent foci . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) . E Amorphous electron - dense structures containing microtubules are formed in RBM14 - depleted cells ( see also Supplementary Fig S5I ) . Electron micrographs from U2OS cells depleted of RBM14 . Squares indicate the ectopic electron - dense structures , whereas circles indicate the pre - existing centrioles . Scale bar , 500 nm and 200 nm ( magnified views ) . Average diameter of pre - existing centrioles and the amorphous electron - dense structures are 225 ± 8 nm ( n = 10 ) and 130 + 11 nm ( n = 16 ) , respectively . Insets show magnified views of the squares . Note that microtubules ( black arrowheads ) are observed within the electron - dense structures ( white arrowheads ) . F Histograms represent frequency of the cells with ectopic foci of GFP - CPAP in the indicated conditions . U2OS cells expressing GFP - CPAP were fixed and stained 24 h after RNAi treatment and induction of GFP - CPAP . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . G U2OS cells expressing GFP - CPAP ( green ) and treated with RBM14 siRNA were analyzed using CLEM . Separate z - plane images of the cell with GFP - CPAP foci and the threefold magnified images are aligned from bottom to top along z - axis ( left LM panels ) . White broken lines represent cell shapes , and blue broken lines represent nuclear shapes . Corresponding regions of EM images are shown on the right . White squares ( a - d ) represent the region around GFP - CPAP foci . Note that other than pre - existing centrioles ( a , 1 - 4 ) , ectopic small CPAP foci are recognizable as amorphous electron - dense structures ( b - d ) . One of pre - existing centrioles ( 1 ) is elongated because of exogenous GFP - CPAP expression . Scale bar , 10 μm ( LM ) , 2 μm ( EM , low magnified image ) , 500 nm [ magnified images of ( a ) and ( d ) ) , 200 nm ( magnified images of ( b ) and ( 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 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 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 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 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

Figure 4A U2OS cells treated with control siRNA, RBM14 siRNA alone or RBM14 + HsSAS-6 siRNAs were stained with antibodies against centrin-2 (green) and HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). n.s., not significant (one-tailed t-test).B Live imaging of cycling RBM14-depleted HeLa cells expressing GFP-centrin1 (green) and RFP-H2B (magenta). Insets show approximately 1.5-fold magnified images of fluorescent foci. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of excess centrin foci formation. Quantification of the number and total signal intensity of centrin foci in HeLa cells treated with control or RBM14 siRNA over time. Means ± SEM are shown (n = 5).C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase. Time-lapse recording across the cell cycle of HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the metaphase onset.D Ectopic formation of centriolar protein complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment. Insets show threefold magnified images of fluorescent foci. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).E Amorphous electron-dense structures containing microtubules are formed in RBM14-depleted cells (see also Supplementary Fig S5I). Electron micrographs from U2OS cells depleted of RBM14. Squares indicate the ectopic electron-dense structures, whereas circles indicate the pre-existing centrioles. Scale bar, 500 nm and 200 nm (magnified views). Average diameter of pre-existing centrioles and the amorphous electron-dense structures are 225 ± 8 nm (n = 10) and 130 + 11 nm (n = 16), respectively. Insets show magnified views of the squares. Note that microtubules (black arrowheads) are observed within the electron-dense structures (white arrowheads).F Histograms represent frequency of the cells with ectopic foci of GFP-CPAP in the indicated conditions. U2OS cells expressing GFP-CPAP were fixed and stained 24 h after RNAi treatment and induction of GFP-CPAP. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test).G U2OS cells expressing GFP-CPAP (green) and treated with RBM14 siRNA were analyzed using CLEM. Separate z-plane images of the cell with GFP-CPAP foci and the threefold magnified images are aligned from bottom to top along z-axis (left LM panels). White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. Corresponding regions of EM images are shown on the right. White squares (a-d) represent the region around GFP-CPAP foci. Note that other than pre-existing centrioles (a, 1-4), ectopic small CPAP foci are recognizable as amorphous electron-dense structures (b-d). One of pre-existing centrioles (1) is elongated because of exogenous GFP-CPAP expression. Scale bar, 10 μm (LM), 2 μm (EM, low magnified image), 500 nm [magnified images of (a) and (d)), 200 nm (magnified images of (b) and (c)].


Figure 5B - E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold - treated for 30 min , followed by 30 - 60 min incubation at 37 ° C and stained with antibodies against centrin ( magenta in ( B ) and ( D ) ) or γ - tubulin ( magenta in ( E ) ) as well as α - tubulin ( green ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point . Values are mean percentages ± standard error of mean ( SEM ) from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 , * * P < 0 . 01 ( one - tailed t - 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

Figure 5B-E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30-60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).


Figure 6A U2OS cells treated with RBM14 siRNA and stained with antibodies against centrin - 2 ( green ) as well as HsSAS - 6 ( magenta ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent the frequency of mitotic cells with the indicated phenotype . Values are mean percentages ± SEM from three independent experiments ( n = 60 for each condition ) . B Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were stained with antibodies against HsSAS - 6 as well as centrin - 2 . Immunofluorescence micrographs are shown in Supplementary Fig S6D . When we counted HsSAS - 6 foci in this paper , we only counted cells before metaphase / anaphase transition considering the disappearance of HsSAS - 6 from anaphase in mitosis . Histograms represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - tailed t - test ) . C U2OS cells treated with the indicated siRNAs were stained with anti - α - tubulin antibodies and Hoechst 33258 . Histograms represent frequency of mitotic cells with the multipolar spindle in the indicated conditions . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - tailed t - test ) . D RBM14 - depleted HeLa cells expressing GFP - centrin ( green ) were stained with antibodies against CP110 ( magenta ) and HsSAS - 6 ( blue ) . Scale bar , 1 μm . A representative stained procentriole - like structure as well as endogenous centrioles is shown together with a schematic . Histograms represent length between the centers of the foci of the indicated proteins . Values are mean percentages ± SEM from seven individual cells . E , F Ectopic DsRed - centrin - 2 foci in RBM14 - depleted cells recruit HsSAS - 6 - GFP . ( E ) Live imaging of RBM14 - depleted U2OS cells expressing HsSAS - 6 - GFP ( green ) and DsRed - centrin - 2 ( magenta ) . Time is denoted in hh : min , and scale bar is 10 μm . Time zero corresponds to the onset of recording . Pre - existing HsSAS - 6 - GFP foci ( white arrowheads ) and ectopic HsSAS - 6 - GFP focus ( red arrowhead ) were traced throughout the time - lapse recording . White broken lines represent cell shapes , and blue broken lines represent nuclear shapes . ( F ) Histograms represent the percentages of ectopic DsRed - centrin - 2 foci with HsSAS - 6 - GFP recruitment ( n = 69 foci for control , and n = 67 foci for RBM14 siRNA ) . G Histograms represent frequency of mitotic cells with excess foci of HsSAS - 6 in the indicated conditions . In this experiment , cells were treated with double amount of RBM14 siRNA ( 20 pmol ) compared to that in the other experiments . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - tailed t - test ) . H HeLa cells expressing GFP - centrin ( green ) and RFP - H2B ( magenta ) and treated with RBM14 siRNA were analyzed using Live CLEM . Pre - existing GFP - centrin foci ( white arrowheads ) and ectopic GFP - centrin foci ( red arrowheads ) were traced throughout the time - lapse recording . Z - stacked confocal images spanning the entire height of the cells ( < 30 μm ) are shown ( upper panels ) . Time is denoted in hh : min . Time zero corresponds to the onset of ectopic formation of GFP - centrin foci . The cells fixed after live imaging were also analyzed for confirming the local correlation between LM and EM images ( lower panels ) . Red square ( ectopically formed GFP - centrin foci ) represents the region corresponding to that of the EM images acquired from serial sections . Note that ectopic GFP - centrin foci that form a spindle pole include not only electron - dense structures ( 1 , 3 ) but also a morphologically recognizable centriole - like structure ( 2 ) . Scale bars , 10 μm ( live imaging ) and 500 nm ( EM ) .

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 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 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 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 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

Figure 6A U2OS cells treated with RBM14 siRNA and stained with antibodies against centrin-2 (green) as well as HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the frequency of mitotic cells with the indicated phenotype. Values are mean percentages ± SEM from three independent experiments (n = 60 for each condition).B Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were stained with antibodies against HsSAS-6 as well as centrin-2. Immunofluorescence micrographs are shown in Supplementary Fig S6D. When we counted HsSAS-6 foci in this paper, we only counted cells before metaphase/anaphase transition considering the disappearance of HsSAS-6 from anaphase in mitosis. Histograms represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).C U2OS cells treated with the indicated siRNAs were stained with anti-α-tubulin antibodies and Hoechst 33258. Histograms represent frequency of mitotic cells with the multipolar spindle in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).D RBM14-depleted HeLa cells expressing GFP-centrin (green) were stained with antibodies against CP110 (magenta) and HsSAS-6 (blue). Scale bar, 1 μm. A representative stained procentriole-like structure as well as endogenous centrioles is shown together with a schematic. Histograms represent length between the centers of the foci of the indicated proteins. Values are mean percentages ± SEM from seven individual cells.E, F Ectopic DsRed-centrin-2 foci in RBM14-depleted cells recruit HsSAS-6-GFP. (E) Live imaging of RBM14-depleted U2OS cells expressing HsSAS-6-GFP (green) and DsRed-centrin-2 (magenta). Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of recording. Pre-existing HsSAS-6-GFP foci (white arrowheads) and ectopic HsSAS-6-GFP focus (red arrowhead) were traced throughout the time-lapse recording. White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. (F) Histograms represent the percentages of ectopic DsRed-centrin-2 foci with HsSAS-6-GFP recruitment (n = 69 foci for control, and n = 67 foci for RBM14 siRNA).G Histograms represent frequency of mitotic cells with excess foci of HsSAS-6 in the indicated conditions. In this experiment, cells were treated with double amount of RBM14 siRNA (20 pmol) compared to that in the other experiments. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).H HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA were analyzed using Live CLEM. Pre-existing GFP-centrin foci (white arrowheads) and ectopic GFP-centrin foci (red arrowheads) were traced throughout the time-lapse recording. Z-stacked confocal images spanning the entire height of the cells (< 30 μm) are shown (upper panels). Time is denoted in hh:min. Time zero corresponds to the onset of ectopic formation of GFP-centrin foci. The cells fixed after live imaging were also analyzed for confirming the local correlation between LM and EM images (lower panels). Red square (ectopically formed GFP-centrin foci) represents the region corresponding to that of the EM images acquired from serial sections. Note that ectopic GFP-centrin foci that form a spindle pole include not only electron-dense structures (1, 3) but also a morphologically recognizable centriole-like structure (2). Scale bars, 10 μm (live imaging) and 500 nm (EM).


Figure 1 ( E ) Confirmation of PARylated proteins with an in vivo PARylation assay . FLAG - tagged candidate proteins were expressed in protoplasts , and immunoprecipitated by α - FLAG antibody after feeding protoplasts with 32P - NAD + . The immunoprecipitated proteins were separated in 10 % SDS - PAGE and detected by autoradiography ( top panel ) . The input of FLAG - tagged proteins is shown in an α - FLAGimmunoblot ( middle panel ) , and the protein loading control is shown by Ponceau S staining for RuBisCo ( RBC ) ( bottom panel ) . The experiments were repeated three times with similar results .

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Figure 1(E) Confirmation of PARylated proteins with an in vivo PARylation assay. FLAG-tagged candidate proteins were expressed in protoplasts, and immunoprecipitated by α-FLAG antibody after feeding protoplasts with 32P-NAD+. The immunoprecipitated proteins were separated in 10% SDS-PAGE and detected by autoradiography (top panel). The input of FLAG-tagged proteins is shown in an α-FLAGimmunoblot (middle panel), and the protein loading control is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel). The experiments were repeated three times with similar results.


Figure 2 ( B ) The DDL transcripts and growth phenotype of ddl - 6 and ddl - 7 . DDL transcripts were detected in fully expanded leaves of four - week - old plants by qRT - PCR ( left panel ) . The expression of DDL in WT Col - 0 was set as 1 . Plants grown under 12 - hr light / 12 - hr dark condition for 4 weeks are shown ( right panel ) . Bar = 1cm . ( C ) ddl - 6 and ddl - 7 are more susceptible to Pst DC3000 infections . Leaves of four - week - old plants were inoculated with Pst DC3000 at OD600 = 5x10 - 4 . Bacterial numbers were counted at 0 and 3 dpi . Leaf pictures were taken at 3 dpi . ( D ) The ddl - 1 mutant in the Ws background is more susceptible to Pst DC3000 infection . ( E ) ddl - 6 is more susceptible to Psminfection . Leaves of four - week - old plants were inoculated with Psmat OD600 = 5x10 - 4 . ( F ) Increased bacterial growth of Pst DC3000 hrcC in ddl - 6 . Leaves of four - week - old plants were inoculated with Pst DC3000 hrcC at OD600 = 5x10 - 4 . ( G ) Increased bacterial growth of Psp in ddl - 6 . Leaves of four - week - old plants were inoculated with Psp at OD600 = 5x10 - 4 . ( H ) Complementation of DDL in disease resistance . Two independent T3 homozygous lines carrying pDDL : : DDL - FLAG ( W7 and W18 ) in the ddl - 6 background were assayed for the susceptibility to Pst DC3000 .

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Figure 2(B) The DDL transcripts and growth phenotype of ddl-6 and ddl-7. DDL transcripts were detected in fully expanded leaves of four-week-old plants by qRT-PCR (left panel). The expression of DDL in WT Col-0 was set as 1. Plants grown under 12-hr light/12-hr dark condition for 4 weeks are shown (right panel). Bar=1cm.(C) ddl-6 and ddl-7 are more susceptible to Pst DC3000 infections. Leaves of four-week-old plants were inoculated with Pst DC3000 at OD600=5x10-4. Bacterial numbers were counted at 0 and 3 dpi. Leaf pictures were taken at 3 dpi.(D) The ddl-1 mutant in the Ws background is more susceptible to Pst DC3000 infection.(E) ddl-6 is more susceptible to Psminfection. Leaves of four-week-old plants were inoculated with Psmat OD600=5x10-4.(F) Increased bacterial growth of Pst DC3000 hrcC in ddl-6. Leaves of four-week-old plants were inoculated with Pst DC3000 hrcC at OD600=5x10-4.(G) Increased bacterial growth of Psp in ddl-6. Leaves of four-week-old plants were inoculated with Psp at OD600=5x10-4.(H) Complementation of DDL in disease resistance. Two independent T3 homozygous lines carrying pDDL::DDL-FLAG (W7 and W18) in the ddl-6 background were assayed for the susceptibility to Pst DC3000.


Figure 3 ( A ) flg22 - induced callose deposition is reduced in ddl - 6 and parp1 , 2 . Leaves of four - week - old plants were inoculated with 0 . 5 µM flg22 for 20 hr or hrcC at OD600 = 0 . 2 for 24 hr and stained with aniline blue . Callose deposits were visualized under UV light and quantified by Image J . The data are shown as mean ± SE ( n = 12 ) . Bar = 100 µm . ( B ) The hrcC - mediated induction of some late responsive genes is compromised in the ddl - 6 and parp1 , 2 mutants . Fully expanded leaves of four - week - old plants were hand - inoculated with hrcC ( OD600 = 0 . 5 ) , and collected at 0 , 3 and 24 hpi for qRT - PCR . The data are shown as mean ± SE from three independent repeats . ( C ) Pst DC3000 - induced PR1and PR5transcripts are blocked in ddl - 6 and parp1 , 2 . Fully expanded leaves of four - week - old plants were inoculated with Pst DC3000 ( OD600 = 0 . 01 ) , and collected at 0 , 8 and 24 hpi for qRT - PCR . The data are shown as mean ± SE from three independent repeats . ( D ) flg22 - induced MAPK activation in Col - 0 and ddl - 6 . Ten - day - old seedlings were treated with 100 nM flg22 and collected at the indicted time points . MAPK activation was analyzed by an immunoblot ( IB ) with - pERK antibody ( top panel ) , and the protein loading is shown by Ponceau S staining for RuBisCo ( RBC ) ( bottom panel ) . ( E ) flg22 - induced ROS in Col - 0 and ddl - 6 . Leaf discs from four - week - old plants were assayed for ROS production upon 100 nM flg22 treatment over 30 min . The data are show as mean ± SE ( n = 24 ) .

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Figure 3(A) flg22-induced callose deposition is reduced in ddl-6 and parp1,2. Leaves of four-week-old plants were inoculated with 0.5 µM flg22 for 20 hr or hrcC at OD600=0.2 for 24 hr and stained with aniline blue. Callose deposits were visualized under UV light and quantified by Image J. The data are shown as mean ± SE (n=12). Bar=100 µm.(B) The hrcC-mediated induction of some late responsive genes is compromised in the ddl-6 and parp1,2 mutants. Fully expanded leaves of four-week-old plants were hand-inoculated with hrcC(OD600=0.5), and collected at 0, 3 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats.(C) Pst DC3000-induced PR1and PR5transcripts are blocked in ddl-6 and parp1,2. Fully expanded leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, 8 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats.(D) flg22-induced MAPK activation in Col-0 and ddl-6. Ten-day-old seedlings were treated with 100 nM flg22 and collected at the indicted time points. MAPK activation was analyzed by an immunoblot (IB) with -pERK antibody (top panel), and the protein loading is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel).(E) flg22-induced ROS in Col-0 and ddl-6. Leaf discs from four-week-old plants were assayed for ROS production upon 100 nM flg22 treatment over 30 min. The data are show as mean± SE (n=24).


Figure 4 ( A ) PARP2 and DDL association in Arabidopsis protoplasts . PARP2 - HA and DDL - FLAG were transiently expressed in Col - 0protoplasts , immunoprecipitated with α - FLAG antibody ( IP : - FLAG ) , and immunoblotted with α - HA ( IB : α - HA ) or α - FLAG antibody ( IB : - FLAG ) ( top two panels ) . The protein inputs are shown with immunoblotting before immunoprecipitation ( bottom two panels ) . Protoplasts were treated without or with 100 nM flg22 for 30 min . ( B ) PARP2 and DDL association in N . benthamiana . PARP2 - FLAG and DDL - HA were transiently co - expressed in leaves of N . benthamiana . The samples were collected at 2 dpi for Co - IP assay with 100nM flg22 treatment for 1 hr . ( C ) PARP2 and DDL association with BiFC assay . Different combinations of constructs were transiently expressed in Arabidopsis Col - 0 protoplasts and the fluorescence signals were observed with a confocal microscope . An NLS - RFP construct was co - transfected to show the nuclear signals . Bar = 10 µm . ( D ) PARP2 and DDL interaction in vitro . GST - PARP2 protein immobilized on glutathione beads was used to pull - downHIS - SUMO - DDL - HA proteins ( left panels ) . A PARylation reaction was performed prior to the pull - down assay ( the third lane of left panels ) . The PARylated PARP2 and DDL were detected with α - PAR antibody ( right panels ) . ( E ) In vitro PARylation of DDL by PARP2 . Immunoprecipitated DDL - FLAG proteins from Arabidopsis protoplasts were incubated with GST - PARP2 in a PARylation reaction containing Biotin - NAD + . The reactions in lane 4 and 5 contained 1 mM 3 - AB or GST - PARG1 respectively . PARylated proteins were detected by streptavidin - HRP antibody ( Strep ) , which recognizes Biotin - labelled PAR .

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 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 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 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

Figure 4(A) PARP2 and DDL association in Arabidopsis protoplasts. PARP2-HA and DDL-FLAG were transiently expressed in Col-0protoplasts, immunoprecipitated with α-FLAG antibody (IP: -FLAG), and immunoblotted with α-HA (IB: α-HA) or α-FLAG antibody (IB: -FLAG) (top two panels). The protein inputs are shown with immunoblotting before immunoprecipitation (bottom two panels). Protoplasts were treated without or with 100 nM flg22 for 30 min.(B) PARP2 and DDL association in N. benthamiana. PARP2-FLAG and DDL-HA were transiently co-expressed in leaves of N. benthamiana. The samples were collected at 2 dpi for Co-IP assay with 100nM flg22 treatment for 1 hr.(C) PARP2 and DDL association with BiFC assay. Different combinations of constructs were transiently expressed in Arabidopsis Col-0 protoplasts and the fluorescence signals were observed with a confocal microscope. An NLS-RFP construct was co-transfected to show the nuclear signals. Bar=10 µm.(D) PARP2 and DDL interaction in vitro. GST-PARP2 protein immobilized on glutathione beads was used to pull-downHIS-SUMO-DDL-HA proteins (left panels). A PARylation reaction was performed prior to the pull-down assay (the third lane of left panels). The PARylated PARP2 and DDL were detected with α-PAR antibody (right panels).(E) In vitro PARylation of DDL by PARP2. Immunoprecipitated DDL-FLAG proteins from Arabidopsis protoplasts were incubated with GST-PARP2 in a PARylation reaction containing Biotin-NAD+. The reactions in lane 4 and 5 contained 1 mM 3-AB or GST-PARG1 respectively. PARylated proteins were detected by streptavidin-HRP antibody (Strep), which recognizes Biotin-labelled PAR.


Figure 5 ( A ) flg22 stimulates DDL PARylation . DDL - FLAG was transiently expressed in Arabidopsis protoplasts fed with 32P - NAD + . Protoplasts were treated without or with 100 nM flg22 for 30 min . PARylated DDL was immunoprecipitated with α - FLAG affinity beads , separated on 10 % SDS - PAGE and visualized by autoradiography . PAR ( DDL ) indicates PARylated DDL proteins . A representative result from three repeats is shown . ( B - C ) DDL PARylation is reduced in parp1 , parp2 and parp1 , 2 mutants . Protoplasts isolated from parp1or parp2 single mutants ( B ) , or parp1 , 2 double mutants ( C ) were used for in vivo PARylation assay with 32P - NAD + . Protoplasts were treated with 100 nM flg22 for 30 min . The experiments were repeated twice with similar results .

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Figure 5(A) flg22 stimulates DDL PARylation. DDL-FLAG was transiently expressed in Arabidopsis protoplasts fed with 32P-NAD+. Protoplasts were treated without or with 100 nM flg22 for 30 min. PARylated DDL was immunoprecipitated with α-FLAG affinity beads, separated on 10% SDS-PAGE and visualized by autoradiography. PAR(DDL) indicates PARylated DDL proteins. A representative result from three repeats is shown.(B-C) DDL PARylation is reduced in parp1, parp2 and parp1,2 mutants. Protoplasts isolated from parp1or parp2 single mutants (B), or parp1,2 double mutants (C) were used for in vivo PARylation assay with 32P-NAD+. Protoplasts were treated with 100 nM flg22 for 30 min. The experiments were repeated twice with similar results.


Figure 6 ( A ) DDL - N but not DDL - C associates with PARP2 . FLAG - tagged DDL - N or DDL - C was co - expressed with PARP2 - HA in protoplasts for Co - IP assay . Protoplasts were treated without or with 100 nM flg22 for 30 min . ( B ) in vivo PARylation of DDL - N or DDL - C in protoplasts fed with 32P - NAD + . PARylation was visualized with autoradiography . ( C ) In vitro PARylation of DDL - N or DDL - C . FLAG - tagged DDL - N or DDL - C was expressed and immunoprecipitated from Arabidopsis protoplasts and subjected to in vitro PARylation by GST - PARP2 with Biotin - NAD + . PARylated proteins were detected by streptavidin - HRP antibody . ( F ) DDL12E mutants substantially diminish its PARylation in vivo . In vivo PARylation of DDL12E1 or DDL12E2 was performed using Arabidopsis protoplasts fed with 32P - NAD + . ( G ) Reduced association of DDL12E1 with PARP2 in protoplasts . Protoplasts were treated with 100 nm flg22 for 30 min before Co - IP assay .

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Figure 6(A) DDL-N but not DDL-C associates with PARP2. FLAG-tagged DDL-N or DDL-C was co-expressed with PARP2-HA in protoplasts for Co-IP assay. Protoplasts were treated without or with 100 nM flg22 for 30 min.(B) in vivo PARylation of DDL-N or DDL-C in protoplasts fed with 32P-NAD+. PARylation was visualized with autoradiography.(C) In vitro PARylation of DDL-N or DDL-C. FLAG-tagged DDL-N or DDL-C was expressed and immunoprecipitated from Arabidopsis protoplasts and subjected to in vitro PARylation by GST-PARP2 with Biotin-NAD+. PARylated proteins were detected by streptavidin-HRP antibody.(F) DDL12E mutants substantially diminish its PARylation in vivo. In vivo PARylation of DDL12E1 or DDL12E2 was performed using Arabidopsis protoplasts fed with 32P-NAD+.(G) Reduced association of DDL12E1 with PARP2 in protoplasts. Protoplasts were treated with 100 nm flg22 for 30 min before Co-IP assay.


Figure 7 ( A ) Phenotype of plants complemented with WT DDL or DDL12E1 . Four - week - old plants of two independent transgenic lines of pDDL : : DDL - FLAG ( W7 and W18 ) or pDDL : : DDL12E1 - FLAG ( E11 and E15 ) . The expression of transgene was determined by an α - FLAGimmunoblot ( bottom ) . ( A ) Phenotype of plants complemented with WT DDL or DDL12E1 . Four - week - old plants of two independent transgenic lines of pDDL : : DDL - FLAG ( W7 and W18 ) or pDDL : : DDL12E1 - FLAG ( E11 and E15 ) were photographed . Bar = 1cm . ( B ) Susceptibility of complementation lines to Pst DC3000 infections . Four - week - old transgenic plants were inoculated with Pst DC3000 ( OD600 = 5x10 - 4 ) and the bacterial number was determined at 0 and 3 dpi . ( C ) PR1 and PR5 induction in transgenic lines . Leaves of four - week - old plants were inoculated with Pst DC3000 ( OD600 = 0 . 01 ) , and collected at 0 , and 24 hpi for qRT - PCR analysis . The data in B and C are shown as mean ± SD ( n = 3 ) from three independent repeats with one - way ANOVA analysis and Tukey test ( p < 0 . 05 ) . Different letters indicate significant differences . ( D ) flg22 - induced callose deposition in transgenic lines . Leaves of four - week - old plants were inoculated with 500 nM flg22 , and callose deposits were stained with aniline blue at 20 hpi . Number of callose deposits was quantified with Image J ( mean ± SE , n = 10 ) . All experiments were repeated three times with similar results .

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 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 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 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

Figure 7(A) Phenotype of plants complemented with WT DDL or DDL12E1. Four-week-old plants of two independent transgenic lines of pDDL::DDL-FLAG (W7 and W18) or pDDL::DDL12E1-FLAG(E11 and E15). The expression of transgene was determined by an α-FLAGimmunoblot (bottom).(A) Phenotype of plants complemented with WT DDL or DDL12E1. Four-week-old plants of two independent transgenic lines of pDDL::DDL-FLAG (W7 and W18) or pDDL::DDL12E1-FLAG(E11 and E15) were photographed. Bar=1cm.(B) Susceptibility of complementation lines to Pst DC3000 infections. Four-week-old transgenic plants were inoculated with Pst DC3000 (OD600=5x10-4) and the bacterial number was determined at 0 and 3 dpi.(C) PR1 and PR5 induction in transgenic lines. Leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, and 24 hpi for qRT-PCR analysis. The data in B and C are shown as mean ± SD (n=3) from three independent repeats with one-way ANOVA analysis and Tukey test (p<0.05). Different letters indicate significant differences.(D) flg22-induced callose deposition in transgenic lines. Leaves of four-week-old plants were inoculated with 500 nM flg22, and callose deposits were stained with aniline blue at 20 hpi. Number of callose deposits was quantified with Image J (mean ± SE, n=10). All experiments were repeated three times with similar results.


Figure 1F , G Immunoblot analysis of ST3GAL5 in ( F ) NMuMG and ( G ) A549 - VIM - RFP cells treated with vehicle control or TGF - β for the indicated time . Vinculin and GAPDH : loading controls .

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Figure 1F, G Immunoblot analysis of ST3GAL5 in (F) NMuMG and (G) A549-VIM-RFP cells treated with vehicle control or TGF-β for the indicated time. Vinculin and GAPDH: loading controls.


Figure 2Immunoblot analysis of p - SMAD2 and total SMAD2 ( t - SMAD2 ) in NMuMG cells with or without Ugcg deficiency and treated with vehicle control or TGF - β for 1 h . Tubulin : loading control . Immunoblot analysis of E - cadherin and N - cadherin in Ugcg KO NMuMG or control NMuMG cells treated with vehicle control or TGF - β for 48 h . Tubulin : loading control .

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Figure 2Immunoblot analysis of p-SMAD2 and total SMAD2 (t-SMAD2) in NMuMG cells with or without Ugcg deficiency and treated with vehicle control or TGF-β for 1 h. Tubulin: loading control.Immunoblot analysis of E-cadherin and N-cadherin in Ugcg KO NMuMG or control NMuMG cells treated with vehicle control or TGF-β for 48 h. Tubulin: loading control.


Figure 3Immunoblot analysis of p - SMAD and t - SMAD2 in A549 - VIM - RFP cells pretreated with eliglustat for 4 or 6 days and then treated with vehicle control or TGF - β for 1 h . GAPDH : loading control . Quantification of the p - SMAD2 level in A549 - VIM - RFP cells as shown in ( A ) . The levels were normalized to that of GAPDH , and fold changes were then further normalized to the level of p - SMAD2 in control cells without TGF - β treatment . Immunoblot analysis of the epithelial marker E - cadherin and mesenchymal markers N - cadherin and vimentin in A549 - VIM - RFP cells treated with eliglustat for 4 or 6 days and / or with vehicle control and / or TGF - β for 2 days . Tubulin : loading control . mCherry - labeled A549 cells were pretreated with eliglustat ( 2 μM ) for 4 days and were then injected into ducts of Cuvier of zebrafish embryos . Representative images with magnified regions ( outlined with dotted squares ) of extravasated cells were acquired 4 days after injection by confocal microscopy . SB group zebrafish were treated with the inhibitor SB505124 ( 1 μM ) in the egg water together with eliglustat for 4 days after injection with daily refreshment of the treatments . Quantification of the number of extravasated cell clusters from 28 embryos per group .

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

Figure 3Immunoblot analysis of p-SMAD and t-SMAD2 in A549-VIM-RFP cells pretreated with eliglustat for 4 or 6 days and then treated with vehicle control or TGF-β for 1 h. GAPDH: loading control. Quantification of the p-SMAD2 level in A549-VIM-RFP cells as shown in (A). The levels were normalized to that of GAPDH, and fold changes were then further normalized to the level of p-SMAD2 in control cells without TGF-β treatment.Immunoblot analysis of the epithelial marker E-cadherin and mesenchymal markers N-cadherin and vimentin in A549-VIM-RFP cells treated with eliglustat for 4 or 6 days and/or with vehicle control and/or TGF-β for 2 days. Tubulin: loading control.mCherry-labeled A549 cells were pretreated with eliglustat (2 μM) for 4 days and were then injected into ducts of Cuvier of zebrafish embryos. Representative images with magnified regions (outlined with dotted squares) of extravasated cells were acquired 4 days after injection by confocal microscopy. SB group zebrafish were treated with the inhibitor SB505124 (1 μM) in the egg water together with eliglustat for 4 days after injection with daily refreshment of the treatments.Quantification of the number of extravasated cell clusters from 28 embryos per group.