( C ) of hepatic glycogen in vehicle ( n = 6 ) - 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 : values are shown as averages ± S . E . M .

O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 densitometric of PYGL in livers of WT 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 : values are shown as averages ± S . E . M .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(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 of liver samples harvested from WT AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bar : 900 nm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(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.


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

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O B-ORGANISM O O O O

(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).


( A ) 293T were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one acid was eliminated cultured for 8 h . Then , WB was used to measure p - AMPK , p - ACC , p - Raptor total AMPK , ACC , Raptor expression . " Nor " = normal medium . Actin served as loading control . red text " - Cys " indicates that cystine deficiency most significantly activates AMPK its substrates .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O

(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 of p - AMPK , p - ACC , p - Raptor total AMPK , ACC , Raptor in 293T 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 loading control .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O

(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 cultured with cystine - deficient medium or complete medium for 8 h were harvested subjected to immunoprecipitation with anti - AMPKα , followed by WB with anti - AMPKα anti - CaMKK2 .

O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O

(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.


( E ) WB of p - AMPK , p - ACC , p - Raptor total AMPK ACC , Raptor expression in 293T transfected with shRNAs targeting CARS that were further treated with cystine - deficient medium for 8 h . Actin served as loading control .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O

(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 of p - AMPK total AMPK expression in CARS - overexpressing 293T RCC4 that were treated with 1 µg / ml STO - 609 or DMSO for 8 h . Actin served as loading control .

O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O

(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.


( D , E ) WB of p - AMPK , p - ACC , p - Raptor total AMPK , ACC , Raptor , AMPKγ2 expression in 293T 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 loading control .

O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O

(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 were transfected with HA - AMPKγ2 alone or with Flag - CARS for 48 h then cultured with cystine - deficient medium or complete medium for 8 h . lysates were immunoprecipitated with anti - Flag , a WB was performed .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY O O O

(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.


( B ) level was determined in 293T with same treatment conditions as in Figure 4A . Data are presented as mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - Student ' s t - ] , compared with indicated groups .

O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) crystal violet ( C ) apoptosis rate ( D ) were performed in 293T transfected with shRNAs targeting NTC , CARS , CaMKK2 or AMPKγ2 further treated with cystine - deficient medium for 24 h with or without 1 mM AICAR . Data are presented as mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - Student ' s t - ] , compared with indicated groups .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 of p - AMPK , total AMPK , CARS , AMPKγ2 , CaMKK2 expression with 786 - O transfected with Flag - EV , Flag - CARS , Flag - CaMKK2 or Flag - AMPKγ2 further treated with cystine - deficient medium for 8 h . Actin served as loading control . Data are presented as mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - Student ' s t - ] , compared with indicated groups .

O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( A ) Immunoblot of HIV - 1 GFP particles ( 2 × 1011 genomes ) produced with lopinavir ( LPV , 0 - 100 nM ) detecting p24 antibody .

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O

(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 on PMA - treated THP - 1 shSAMHD1 . Mean ± SD , n = 3 .

O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O

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 on PMA - treated U87 . Mean ± SD , n = 3 .

O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O

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 transduced for 24 h with LPV - treated HIV - 1 GFP ( 0 . 1 U RT / ml red , 0 . 5 U RT / ml blue ) .

O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O

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 transduced for 24 h with LPV - treated HIV - 1 GFP ( 0 . 1 U RT / ml red , 0 . 5 U RT / ml blue ) . G ) CXCL - 10 in supernatants from D - F ( ELISA ) .

B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O

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 transduced for 24 h with 0 . 2 U RT / ml 30 nM LPV - treated HIV - 1 GFP in presence of DMSO vehicle or 2 μM ruxolitinib . A control was stimulated with 1 ng / ml IFNβ .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O

(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 transduced for 24 h with 0 . 3 U RT / ml LPV - treated HIV - 1 GFP .

O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O

(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 particles ( 2 × 1011 genomes ) produced with LPV ( 0 - 100 nM ) detecting p24 .

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O

(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 ) infection data ( L ) from monocyte - derived macrophages ( MDM ) infected for 24 h with LPV - treated HIV - 1 R9 BaL ( 0 . 2 U RT / ml ) . Data are collated from two donors ( represented by circles squares ) , n = 3 . Horizontal represents median . For experiments in which dose used was normalised by RT activity , number of genome copies was also measured by qPCR of . This gave dose equivalents of within 2 - 3 fold of RT equivalents .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O

(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.


( A ) Immunoblot of HIV - 1 GFP particles ( 2 × 1011 genomes ) with varying proportions of ΔCA - SP1 protease cleavage mutation detecting p24 .

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O

(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 on U87 . Mean ± SD , n = 3 .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O O O O

(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 transduced for 24 h with HIV - 1 GFP ΔCA - SP1 ( 0 . 1 U RT / ml red , 0 . 5 U RT / ml blue ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(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 transduced for 24 h with HIV - 1 GFP ΔCA - SP1 ( 0 . 1 U RT / ml red , 0 . 5 U RT / ml blue ) .

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 transduced for 24 h with HIV - 1 GFP ΔCA - SP1 ( 0 . 1 U RT / ml red , 0 . 5 U RT / ml blue ) . E ) CXCL - 10 in supernatants from ( C , D ) ( ELISA ) .

B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O

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 transduced for 24 h with 6 × 109 genomes / ml ( approx . 0 . 5 U RT / ml ) HIV - 1 GFP ΔCA - SP1 .

O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O

(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 transduced for 24 h with HIV - 1 GFP ΔCA - SP1 ( 0 . 016 - 0 . 2 U RT / ml ) . Data are shown as measurements , representative of 2 repeats .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 presence of DMSO vehicle or 2 μM ruxolitinib .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O

(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 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 0 . 5 U RT / ml ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 presence of DMSO vehicle or 2 μM ruxolitinib .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O

(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.


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 presence of DMSO vehicle or 2 μM ruxolitinib . K ) CXCL - 10 in supernatants from ( J ) ( ELISA ) .

B-CELL_TYPE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O

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).


( A ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O

(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 transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . B ) CXCL - 10 in supernatants from ( A ) ( ELISA ) .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O

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 transduced for 24 h with 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 109 3 × 109 genomes / ml ) in presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O

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 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 1 . 5 × 1010 genomes / ml ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

(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 in supernatants from ( E ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O

(F) CXCL-10 protein in supernatants from (E) (ELISA). Triangles indicate CXCL-10 not detected.


( A ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 lacking STING or MAVS , or a gRNA control ( Ctrl ) transduced for 24 h with HIV - 1 GFP 75 % ΔCA - SP1 ( 0 . 4 U RT / ml ) or stimulated by transfection with either HT - ( 0 . 1 μg / ml ) or poly I : C ( 0 . 5 μg / ml ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(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 lacking cGAS or a matching control ( Ctrl ) stimulated for 24 h with poly I : C ( transfection , 0 . 5 μg / ml ) , HT - ( transfection , 0 . 1 μg / ml ) , LPS ( 50 ng / ml ) or cGAMP ( transfection , 0 . 5 μg / ml )

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

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 lacking cGAS or a matching control ( Ctrl ) transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 2 × 1010 genomes / ml )

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

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 lacking cGAS or a matching control ( Ctrl ) transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 2 × 1010 genomes / ml ) ( C ) . D ) CXCL - 10 in supernatants from ( C ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O

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 lacking STING , MAVS or matching gRNA control ( Ctrl ) transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O

(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 lacking cGAS or matching control ( Ctrl ) transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O

(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 lacking STING , MAVS or matching gRNA control ( Ctrl ) transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . G ) CXCL - 10 in supernatants from ( E ) .

B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

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 lacking cGAS or matching control ( Ctrl ) transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . ( H ) CXCL - 10 in supernatants from ( F ) ( ELISA ) .

B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O

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 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 presence of DMSO vehicle , 2 μM ruxolitinib or 0 . 5 μg / ml H151 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O

(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.


Abrogation - of - restriction in FRhK4 expressing restrictive rhesus TRIM5 . FRhK4 were co - transduced with a fixed dose of HIV - 1 GFP ( 5 × 107 genomes / ml ) increasing doses of HIV - LUC ΔCA - SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values two repeats of 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 O O O O O B-CELL_LINE O O O B-GENEPROD O B-CELL_LINE O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abrogation-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 in FRhK4 expressing restrictive rhesus TRIM5 . FRhK4 were co - transduced with LPV - treated HIV - LUC Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values two repeats of 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 O O O O O B-CELL_LINE O O O B-GENEPROD O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


( A ) Infection data for THP - 1 - IFIT - 1 transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in presence of DMSO vehicle or PF - 74 ( 10 μM ) .

O O O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

(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 transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in presence of DMSO vehicle or PF - 74 ( 10 μM ) . B ) IFIT - 1 reporter activity in supernatant from ( A ) .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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 transduced for 24 h with HIV - 1 GFP ( 0 . 25 - 1 U / ml RT ) in presence of DMSO vehicle or PF - 74 ( 10 μM ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

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 in supernatants of THP - 1 - IFIT - 1 transduced for 24 h with HIV - 1 GFP ( 3 U / ml ) in presence of DMSO vehicle or PF - 74 ( 10 μM ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

(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 transduced for 24 h with HIV - 1 GFP ( 3 U / ml RT ) in presence of DMSO vehicle or PF - 74 ( 10 μM ) ruxolitinib ( Rux , 2 μM ) as indicated .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

(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 lacking cGAS or a matching control ( Ctrl ) transduced for 24 h with HIV - 1 GFP ( 3 U / ml 6 U / ml ) in presence of DMSO vehicle or PF - 74 ( 10 μM ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

(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 lacking MAVS or a matching gRNA control ( Ctrl ) transduced for 24 h with HIV - 1 GFP ( 3 U / ml 6 U / ml ) in presence of DMSO vehicle or PF - 74 ( 10 μM ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

(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).


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

O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-CELL_LINE O B-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Depletion 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.


A U2OS or U2OS expressing FLAG - RBM14 full ( FL , aa1 - 669 ) , N - terminal fragment ( RBM14 [ N ] , aa1 - 150 ) or C - terminal fragment ( RBM14 [ C ] , aa151 - 669 ) 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 with excess centrin foci at spindle poles in each condition . Values are mean percentages ± from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - t - ) .

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A 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 RBM14 could suppress centriole amplification in HU - treated . ( C ) U2OS or U2OS expressing FLAG - RBM14 FL or [ C ] treated with or without HU were stained with antibodies against centrin - 2 ( green ) FLAG ( magenta ) . ( D ) U2OS or U2OS expressing FLAG - RBM14 FL , FLAG - RBM14 - NES or GFP - RBM14 - PACT 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 foci around centrosome . Scale bar , 10 μm . Histograms represent frequency of in interphase with excess centrin foci in each condition . percentages of U2OS with centrosomal localization of RBM14 full - or mutants are shown below immunofluorescence . We counted only that had adequate of FLAG or GFP signals did not find any significant difference in total expression of exogenous RBM14 . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - t - ) . Please note that expression of GFP - RBM14 - PACT are less than those of FLAG - RBM14 FL ( ˜ 0 . 5 - fold ) or FLAG - RBM14 - NES ( ˜ 0 . 7 - fold ) .

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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).


A HeLa immunoprecipitated with control IgG or STIL antibodies . Soluble fractions ( input ) immunoprecipitates ( IPs ) were analyzed by Western blotting using RBM14 , STIL or CPAP antibodies .

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A 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 testing interactions between purified STIL [ N ] ( ˜ 5 μg , aa1 - 1018 ) GST - RBM14 [ N ] or [ C ] . asterisks indicate non - specific bands .

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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 treated with control or RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) STIL ( magenta ) . Insets show approximately twofold magnified views of foci around centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic with excess STIL foci co - localized with centrin foci . Values are mean percentages ± from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 ( one - t - ) .

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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 . GST pull - down experiment was performed as in ( B ) , with purified STIL [ N ] GST - RBM14 [ C ] in presence of indicated amount of purified His - CPAP [ SBD ] , His - tagged STIL - Binding Domain of CPAP . fraction of STIL [ N ] bound to GST - RBM14 [ C ] in such conditions was monitored by Western blotting using STIL antibodies which recognize N - terminal region of STIL . Input materials were analyzed by Western blotting using STIL or CPAP antibodies . precipitated GST - RBM14 [ C ] was analyzed by SDS - PAGE , stained with SimplyBlue ™ Safe ( Invitrogen ) .

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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 GFP - CPAP in presence of FLAG - RBM14 FL or fragments . U2OS 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 , resulting IPs were analyzed by Western blotting using STIL , CPAP or FLAG antibodies . of relative amounts of co - immunoprecipitated GFP - CPAP in each STIL - IP fraction , normalized with amount of STIL precipitated , is shown below panels . Means ± were calculated from three independent experiments . * * P < 0 . 01 ( one - t - ) . Note that we did not find any significant effect of exogenous expression of RBM14 full - or deletion mutants on expression of GFP - CPAP or endogenous STIL in this experiment .

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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 treated with indicated siRNAs were stained with antibodies against centrin - 2 ( green ) C - Nap1 ( magenta ) . Histograms represent frequency of mitotic with excess centrin foci at spindle poles in each condition . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - t - ) . Representative mitotic treated with siRNAs against RBM14 alone , RBM14 + STIL or RBM14 + CPAP are shown below . Insets show approximately twofold magnified views of foci around centrosome . Scale bar , 5 μm .

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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.


A U2OS treated with control siRNA , RBM14 siRNA alone or RBM14 + HsSAS - 6 siRNAs were stained with antibodies against centrin - 2 ( green ) HsSAS - 6 ( magenta ) . is shown in blue . Insets show approximately twofold magnified views of foci around centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic with excess centrin foci in indicated conditions . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . n . s . , not significant ( one - t - ) .

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A 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 expressing GFP - centrin1 ( green ) RFP - H2B ( magenta ) . Insets show approximately 1 . 5 - fold magnified of foci . Time is denoted in hh : min , scale bar is 10 μm . Time zero corresponds to onset of excess centrin foci formation . of number total signal of centrin foci in HeLa treated with control or RBM14 siRNA over time . Means ± are shown ( n = 5 ) .

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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 cycle of HeLa expressing GFP - centrin ( green ) RFP - H2B ( magenta ) treated with RBM14 siRNA . Time is denoted in hh : min , scale bar is 10 μm . Time zero corresponds to metaphase onset .

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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 complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment . Insets show threefold magnified of foci . Scale bar , 5 μm . Histograms represent frequency of mitotic with excess centrin foci in indicated conditions . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - t - ) .

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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 ( see also Supplementary Fig S5I ) . Electron from U2OS depleted of RBM14 . Squares indicate ectopic electron - dense structures , whereas circles indicate pre - existing centrioles . Scale bar , 500 nm 200 nm ( magnified views ) . Average diameter of pre - existing centrioles amorphous electron - dense structures are 225 ± 8 nm ( n = 10 ) 130 + 11 nm ( n = 16 ) , respectively . Insets show magnified views of squares . Note that microtubules ( black arrowheads ) are observed within electron - dense structures ( arrowheads ) .

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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 with ectopic foci of GFP - CPAP in indicated conditions . U2OS expressing GFP - CPAP were fixed stained 24 h after RNAi treatment induction of GFP - CPAP . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - t - ) .

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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 expressing GFP - CPAP ( green ) treated with RBM14 siRNA were analyzed using CLEM . Separate z - plane of with GFP - CPAP foci threefold magnified are aligned from bottom to top along z - axis ( left LM panels ) . broken lines represent shapes , blue broken lines represent shapes . Corresponding regions of EM are shown on right . squares ( a - d ) represent 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 of ( a ) ( d ) ) , 200 nm ( magnified of ( b ) ( c ) ] .

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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)].


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

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B-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).


A U2OS treated with RBM14 siRNA stained with antibodies against centrin - 2 ( green ) as well as HsSAS - 6 ( magenta ) . is shown in blue . Insets show approximately twofold magnified views of foci around centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic with indicated phenotype . Values are mean percentages ± from three independent experiments ( n = 60 for each condition ) .

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A 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 or U2OS treated with siRNA targeting RBM14 were stained with antibodies against HsSAS - 6 as well as centrin - 2 . Immunofluorescence are shown in Supplementary Fig S6D . When we counted HsSAS - 6 foci in this paper , we only counted before metaphase / anaphase transition considering disappearance of HsSAS - 6 from anaphase in mitosis . Histograms represent frequency of mitotic with excess foci of indicated at spindle poles . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - t - ) .

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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 treated with indicated siRNAs were stained with anti - α - tubulin antibodies Hoechst 33258 . Histograms represent frequency of mitotic with multipolar spindle in indicated conditions . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - t - ) .

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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 expressing GFP - centrin ( green ) were stained with antibodies against CP110 ( magenta ) 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 between centers of foci of indicated . Values are mean percentages ± from seven .

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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 recruit HsSAS - 6 - GFP . ( E ) Live imaging of RBM14 - depleted U2OS expressing HsSAS - 6 - GFP ( green ) DsRed - centrin - 2 ( magenta ) . Time is denoted in hh : min , scale bar is 10 μm . Time zero corresponds to onset of recording . Pre - existing HsSAS - 6 - GFP foci ( arrowheads ) ectopic HsSAS - 6 - GFP focus ( red arrowhead ) were traced throughout time - lapse recording . broken lines represent shapes , blue broken lines represent shapes . ( F ) Histograms represent percentages of ectopic DsRed - centrin - 2 foci with HsSAS - 6 - GFP recruitment ( n = 69 foci for control , n = 67 foci for RBM14 siRNA ) .

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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 with excess foci of HsSAS - 6 in indicated conditions . In this experiment , were treated with double amount of RBM14 siRNA ( 20 pmol ) compared to that in other experiments . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - t - ) .

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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 expressing GFP - centrin ( green ) RFP - H2B ( magenta ) treated with RBM14 siRNA were analyzed using Live CLEM . Pre - existing GFP - centrin foci ( arrowheads ) ectopic GFP - centrin foci ( red arrowheads ) were traced throughout time - lapse recording . Z - stacked confocal spanning entire height of ( < 30 μm ) are shown ( upper panels ) . Time is denoted in hh : min . Time zero corresponds to onset of ectopic formation of GFP - centrin foci . fixed after live imaging were also analyzed for confirming local correlation between LM EM ( lower panels ) . Red square ( ectopically formed GFP - centrin foci ) represents region corresponding to that of EM 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 ) 500 nm ( EM ) .

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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).


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

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(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.


( B ) DDL transcripts growth phenotype of ddl - 6 ddl - 7 . DDL transcripts were detected in fully expanded leaves of four - week - old by qRT - PCR ( left panel ) . expression of DDL in WT Col - 0 was set as 1 . grown under 12 - hr / 12 - hr condition for 4 are shown ( right panel ) . Bar = 1cm .

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(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 ddl - 7 are more susceptible to Pst DC3000 infections . Leaves of four - week - old were inoculated with Pst DC3000 at OD600 = 5x10 - 4 . numbers were counted at 0 3 dpi . Leaf pictures were taken at 3 dpi .

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(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 ) ddl - 1 mutant in Ws background is more susceptible to Pst DC3000 infection .

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(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 were inoculated with Psmat OD600 = 5x10 - 4 .

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(E) ddl-6 is more susceptible to Psminfection. Leaves of four-week-old plants were inoculated with Psmat OD600=5x10-4.


( F ) Increased growth of Pst DC3000 hrcC in ddl - 6 . Leaves of four - week - old were inoculated with Pst DC3000 hrcC at OD600 = 5x10 - 4 .

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(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 growth of Psp in ddl - 6 . Leaves of four - week - old were inoculated with Psp at OD600 = 5x10 - 4 .

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(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 W18 ) in ddl - 6 background were assayed for susceptibility to Pst DC3000 .

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(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.


( A ) flg22 - induced callose deposition is reduced in ddl - 6 parp1 , 2 . Leaves of four - week - old were inoculated with 0 . 5 µM flg22 for 20 hr or hrcC at OD600 = 0 . 2 for 24 hr stained with aniline blue . Callose deposits were visualized under UV quantified by Image J . data are shown as mean ± SE ( n = 12 ) . Bar = 100 µm .

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(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 ) hrcC - mediated induction of some late responsive genes is compromised in ddl - 6 parp1 , 2 mutants . Fully expanded leaves of four - week - old were - inoculated with hrcC ( OD600 = 0 . 5 ) , collected at 0 , 3 24 hpi for qRT - PCR . data are shown as mean ± SE from three independent repeats .

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(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 parp1 , 2 . Fully expanded leaves of four - week - old were inoculated with Pst DC3000 ( OD600 = 0 . 01 ) , collected at 0 , 8 24 hpi for qRT - PCR . data are shown as mean ± SE from three independent repeats .

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(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 ddl - 6 . Ten - day - old seedlings were treated with 100 nM flg22 collected at indicted time points . MAPK activation was analyzed by an immunoblot ( IB ) with - pERK antibody ( top panel ) , loading is shown by Ponceau S for RuBisCo ( RBC ) ( bottom panel ) .

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(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 ddl - 6 . Leaf discs from four - week - old were assayed for ROS production upon 100 nM flg22 treatment over 30 min . data are show as mean ± SE ( n = 24 ) .

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(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).


( A ) PARP2 DDL association in Arabidopsis protoplasts . PARP2 - HA DDL - FLAG were transiently expressed in Col - 0protoplasts , immunoprecipitated with α - FLAG antibody ( IP : - FLAG ) , immunoblotted with α - HA ( IB : α - HA ) or α - FLAG antibody ( IB : - FLAG ) ( top two panels ) . inputs are shown with immunoblotting before immunoprecipitation ( bottom two panels ) . Protoplasts were treated without or with 100 nM flg22 for 30 min .

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(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 DDL association in N . benthamiana . PARP2 - FLAG DDL - HA were transiently co - expressed in leaves of N . benthamiana . samples were collected at 2 dpi for Co - IP with 100nM flg22 treatment for 1 hr .

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(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 DDL association with BiFC . Different combinations of constructs were transiently expressed in Arabidopsis Col - 0 protoplasts signals were observed with a confocal microscope . An NLS - RFP construct was co - transfected to show signals . Bar = 10 µm .

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(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 DDL interaction in vitro . GST - PARP2 immobilized on glutathione beads was used to pull - downHIS - SUMO - DDL - HA ( left panels ) . A PARylation reaction was performed prior to pull - down ( third lane of left panels ) . PARylated PARP2 DDL were detected with α - PAR antibody ( right panels ) .

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(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 from Arabidopsis protoplasts were incubated with GST - PARP2 in a PARylation reaction containing Biotin - NAD + . reactions in lane 4 5 contained 1 mM 3 - AB or GST - PARG1 respectively . PARylated were detected by streptavidin - HRP antibody ( Strep ) , which recognizes Biotin - labelled PAR .

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(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.


( 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 visualized by autoradiography . PAR ( DDL ) indicates PARylated DDL . A representative result from three repeats is shown .

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(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 parp1 , 2 mutants . Protoplasts isolated from parp1or parp2 single mutants ( B ) , or parp1 , 2 double mutants ( C ) were used for in vivo PARylation with 32P - NAD + . Protoplasts were treated with 100 nM flg22 for 30 min . experiments were repeated twice with similar results .

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(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.


( 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 . Protoplasts were treated without or with 100 nM flg22 for 30 min .

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(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 .

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(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 immunoprecipitated from Arabidopsis protoplasts subjected to in vitro PARylation by GST - PARP2 with Biotin - NAD + . PARylated were detected by streptavidin - HRP antibody .

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(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 + .

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(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 .

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(G) Reduced association of DDL12E1 with PARP2 in protoplasts. Protoplasts were treated with 100 nm flg22 for 30 min before Co-IP assay.


( A ) Phenotype of complemented with WT DDL or DDL12E1 . Four - week - old of two independent transgenic lines of pDDL : : DDL - FLAG ( W7 W18 ) or pDDL : : DDL12E1 - FLAG ( E11 E15 ) . expression of transgene was determined by an α - FLAGimmunoblot ( bottom ) .

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(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 complemented with WT DDL or DDL12E1 . Four - week - old of two independent transgenic lines of pDDL : : DDL - FLAG ( W7 W18 ) or pDDL : : DDL12E1 - FLAG ( E11 E15 ) were photographed . Bar = 1cm .

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(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 were inoculated with Pst DC3000 ( OD600 = 5x10 - 4 ) number was determined at 0 3 dpi .

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(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 PR5 induction in transgenic lines . Leaves of four - week - old were inoculated with Pst DC3000 ( OD600 = 0 . 01 ) , collected at 0 , 24 hpi for qRT - PCR . data in B C are shown as mean ± SD ( n = 3 ) from three independent repeats with one - way ANOVA Tukey ( p < 0 . 05 ) . Different letters indicate significant differences .

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(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 were inoculated with 500 nM flg22 , callose deposits were stained with aniline blue at 20 hpi . Number of callose deposits was quantified with Image J ( mean ± SE , n = 10 ) . experiments were repeated three times with similar results .

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(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.


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

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F, 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.


Immunoblot of p - SMAD2 total SMAD2 ( t - SMAD2 ) in NMuMG with or without Ugcg deficiency treated with vehicle control or TGF - β for 1 h . Tubulin : loading control .

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Immunoblot 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.