Figure 1B , BrdU incorporation assay for H460 and A549 cells . The mean ± SD represents 6 biological replicates in one experiment . Three independent experiments were performed . * * p < 0 . 01 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test . D , Immunoblot showing the expression of Cleaved - PARP , Cleaved - CASP3 , CASP3 and Actin in H460 and A549 cells . E , H460 and A549 cells were cultured in 5 % Matrigel for 10 days . Representative images of the 3D Matrigel culture are shown in the top panel , and immunofluorescence staining with anti - laminin5 and DAPI is shown in the bottom panel ( laminin5 , red ; DAPI , blue ) . Scale bars , 200 μm . The right panel shows the percentages of colonies with a diameter ≥ 50 μm and colonies with a diameter < 50 μm . The mean ± SD represents 5 visual fields from one experiment . Three independent experiments were performed . * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test . F , The relative cytoplasmic nucleosomes index was measured by detection with the cell death detection ELISA kit for H460 and A549 cells after 24 h under attached or suspended conditions . Immunoblot showing the expression of Cleaved - PARP , Cleaved - CASP3 , CASP3 and Actin in H460 and A549 cells after 24 h under attached or suspended conditions . The mean ± SD represents 4 biological replicates in one experiment . Three independent experiments were performed . ns , nonsignificant , * * p < 0 . 01 , unpaired two - tailed Student ' s t test . G , The indicated cells were allowed to grow in soft agar for 2 weeks , and colonies were counted . The mean ± SD represents 3 biological replicates in one experiment . Three independent experiments were performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , unpaired two - tailed Student ' s t test . J , The volcano plot maps based on RNA - seq data show changes in the gene expression profile in H460 ( C / C ) cells versus H460 ( T / T ) cells .

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

Figure 1B, BrdU incorporation assay for H460 and A549 cells. The mean ± SD represents 6 biological replicates in one experiment. Three independent experiments were performed. **p < 0.01, ****p < 0.0001, unpaired two-tailed Student's t test.D, Immunoblot showing the expression of Cleaved-PARP, Cleaved-CASP3, CASP3 and Actin in H460 and A549 cells.E, H460 and A549 cells were cultured in 5% Matrigel for 10 days. Representative images of the 3D Matrigel culture are shown in the top panel, and immunofluorescence staining with anti-laminin5 and DAPI is shown in the bottom panel (laminin5, red; DAPI, blue). Scale bars, 200 μm. The right panel shows the percentages of colonies with a diameter ≥ 50 μm and colonies with a diameter < 50 μm. The mean ± SD represents 5 visual fields from one experiment. Three independent experiments were performed. ****p < 0.0001, unpaired two-tailed Student's t test.F, The relative cytoplasmic nucleosomes index was measured by detection with the cell death detection ELISA kit for H460 and A549 cells after 24 h under attached or suspended conditions. Immunoblot showing the expression of Cleaved-PARP, Cleaved-CASP3, CASP3 and Actin in H460 and A549 cells after 24 h under attached or suspended conditions. The mean ± SD represents 4 biological replicates in one experiment. Three independent experiments were performed. ns, nonsignificant, **p < 0.01, unpaired two-tailed Student's t test.G, The indicated cells were allowed to grow in soft agar for 2 weeks, and colonies were counted. The mean ± SD represents 3 biological replicates in one experiment. Three independent experiments were performed. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student's t test.J, The volcano plot maps based on RNA-seq data show changes in the gene expression profile in H460 (C/C) cells versus H460 (T/T) cells.


Figure 2A - C , H460 ( T / T ) , H460 ( C / C ) , A549 ( T / T ) and A549 ( C / C ) cells were subcutaneously inoculated into BALB / c nude mice ( n = 5 ) . Primary tumors were resected after 5 weeks ( A ) . The graphs show the mean tumor volumes ( B ) and weight ( C ) . Values shown are means ± SD obtained from animal experiments . * p < 0 . 05 , * * p < 0 . 01 , two - way ANOVA ( B ) and unpaired two - tailed Student ' s t test ( C ) . D , IHC staining for Ki67 in primary tumors from animals . The panels show representative images ( left ) , and the graph shows the quantification of Ki67 - positive cells in the tumor ( right ) . Scale bars , 100 μm . The mean ± SD represents 5 visual fields from each tumor tissue . * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test . F , The scatter plot and the percentage of HLA - I - positive circulating tumor cells ( CTCs ) in the peripheral blood was analyzed by Flow Cytometry , and numerical statistics of the two groups were calculated ( n = 5 ) . Tumor - free mice were used as a negative control to determine the gating . * p < 0 . 05 , * * p < 0 . 01 , unpaired two - tailed Student ' s t test . I , The log - rank ( Mantel - Cox ) test of overall survival of 239 Chinese lung cancer patients based on rs1663689 genotype .

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

Figure 2A-C, H460 (T/T), H460 (C/C), A549 (T/T) and A549 (C/C) cells were subcutaneously inoculated into BALB/c nude mice (n = 5). Primary tumors were resected after 5 weeks (A). The graphs show the mean tumor volumes (B) and weight (C). Values shown are means ± SD obtained from animal experiments. *p < 0.05, **p < 0.01, two-way ANOVA (B) and unpaired two-tailed Student's t test (C).D, IHC staining for Ki67 in primary tumors from animals. The panels show representative images (left), and the graph shows the quantification of Ki67-positive cells in the tumor (right). Scale bars, 100 μm. The mean ± SD represents 5 visual fields from each tumor tissue. ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.F, The scatter plot and the percentage of HLA-I-positive circulating tumor cells (CTCs) in the peripheral blood was analyzed by Flow Cytometry, and numerical statistics of the two groups were calculated (n = 5). Tumor-free mice were used as a negative control to determine the gating. *p < 0.05, **p < 0.01, unpaired two-tailed Student's t test.I, The log-rank (Mantel-Cox) test of overall survival of 239 Chinese lung cancer patients based on rs1663689 genotype.


Figure 3F , The location diagram shows 4C enrichment around the negative control and ADGRG6 probe . Representative images of nuclei stained with DNA FISH probes for SNP rs1666389 ( green ) and negative control ( red ) or ADGRG6 ( red ) in H460 and A549 cells . The numerical points in the figure show the percentage of colocation in seven slides ( with a total of 80 nuclei ) . Arrows point to the colocation signals . Nuclei with green or red positive signal are listed in the counting range . Values shown are means ± SD . Scale bars , 5 μm . ns , nonsignificant , * p < 0 . 05 , * * p < 0 . 01 , unpaired two - tailed Student ' s t test . G , Expression of ADGRG6 at the protein levels in H460 and A549 cells . H , eQTL analysis demonstrating the correlation between the rs1663689 genotype and the expression of ADGRG6 in the clinical patient cohorts . The p value of the linear model was assessed by the Kruskal - Wallis test . Values shown are means ± SD .

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

Figure 3F, The location diagram shows 4C enrichment around the negative control and ADGRG6 probe. Representative images of nuclei stained with DNA FISH probes for SNP rs1666389 (green) and negative control (red) or ADGRG6 (red) in H460 and A549 cells. The numerical points in the figure show the percentage of colocation in seven slides (with a total of 80 nuclei). Arrows point to the colocation signals. Nuclei with green or red positive signal are listed in the counting range. Values shown are means ± SD. Scale bars, 5 μm. ns, nonsignificant, *p < 0.05, **p < 0.01, unpaired two-tailed Student's t test.G, Expression of ADGRG6 at the protein levels in H460 and A549 cells.H, eQTL analysis demonstrating the correlation between the rs1663689 genotype and the expression of ADGRG6 in the clinical patient cohorts. The p value of the linear model was assessed by the Kruskal-Wallis test. Values shown are means ± SD.


Figure 4A , Protein expression of ADGRG6 or Actin after ADGRG6 knockdown in T / T cells and overexpression in C / C cells . Boyden chamber assay ( C ) of ADGRG6 - knockdown T / T cells and ADGRG6 - overexpressing C / C cells . Values shown are means ± SD obtained from one experiment . Scale bars , 200 μm . Three independent experiments were performed . * * p < 0 . 01 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test .

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Figure 4A, Protein expression of ADGRG6 or Actin after ADGRG6 knockdown in T/T cells and overexpression in C/C cells.Boyden chamber assay (C) of ADGRG6-knockdown T/T cells and ADGRG6-overexpressing C/C cells. Values shown are means ± SD obtained from one experiment. Scale bars, 200 μm. Three independent experiments were performed. **p < 0.01, ****p < 0.0001, unpaired two-tailed Student's t test.


Figure 5A , Immunoblot showing the expression of PKA substrate phosphorylation , PKA and Actin in the indicated cells and genotypes and the indicated cells treated with H89 . BrdU incorporation assay ( B ) of the indicated cells after treatment with H89 . Scale bars , 200 μm . Values shown are means ± SD obtained from one experiment . Three independent experiments were performed . ns , nonsignificant , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test . D , Representative images and quantification of patient - derived organoids treated with 20 μM H89 and controls . The mean ± SD represents 5 visual fields from one experiment . Scale bars , 20 μm . ns , nonsignificant , * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test .

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Figure 5A, Immunoblot showing the expression of PKA substrate phosphorylation, PKA and Actin in the indicated cells and genotypes and the indicated cells treated with H89.BrdU incorporation assay (B) of the indicated cells after treatment with H89. Scale bars, 200 μm. Values shown are means ± SD obtained from one experiment. Three independent experiments were performed. ns, nonsignificant, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.D, Representative images and quantification of patient-derived organoids treated with 20 μM H89 and controls. The mean ± SD represents 5 visual fields from one experiment. Scale bars, 20 μm. ns, nonsignificant, ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.


Figure 2A Cell cycle analysis by flow cytometry ( EdU vs . TO - PRO - 3 ) in complete medium ( ENRAD ) or in the absence of A83 - 01 ( 24 hours ) . B Detection of proteins secreted by mouse prostate organoids in culture based on click - chemistry enrichment and mass spectrometry analysis ( n = 4 biological replicates ; data points are shown with crossing line representing mean value ) . C IF analysis for selected markers in mouse prostate tissue and organoid sections ( scale bar = 50 μm ) . D Representative stereoscopic images of mouse prostate organoids cultured in the absence of A83 - 01 , with or without Follistatin ( Fst , 500 ng / mL , 6 days ) , or with or without the Activin A neutralizing antibody MAB3381 ( 1 μg / mL , 6 days ) . Scale bar = 1 mmE Western blot ( WB ) analysis in mouse prostate organoids for selected canonical and non - canonical Tgf - β signalling mediators , and the cell cycle inhibitor p21 ( Activin A , 50 ng / mL ; Tgfb1 , 500 ng / mL ; 24 hours ) . G Representative stereoscopic images of mouse prostate organoids following treatment with Takinib ( Tak1 inhibitor ; 5 μM , 6 days ) , SB203580 ( p38α / β inhibitor ; 10 μM , 6 days ) or the structurally unrelated Ralimetinib ( p38α / β inhibitor ; 1 μM , 7 days ) . Scale bar = 1 mmH Nuclear / Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected signalling mediators in the presence or absence of SB203580 ( p38α / β inhibitor ; 10 μM , 24 hours ) . I Gene set enrichment analysis ( GSEA ) plots displaying significant enrichment for inflammatory response and Tumor Necrosis Factor ( TNF ) signalling in mouse prostate organoids cultured without A83 - 01 ( 24 hours ) versus complete medium ( ENRAD ) . J Nuclear / Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected cell cycle regulators , in the presence or absence of SB203580 ( p38α / β inhibitor ; 10 μM , 24 or 48 hours ) .

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

Figure 2A Cell cycle analysis by flow cytometry (EdU vs. TO-PRO-3) in complete medium (ENRAD) or in the absence of A83-01 (24 hours).B Detection of proteins secreted by mouse prostate organoids in culture based on click-chemistry enrichment and mass spectrometry analysis (n=4 biological replicates; data points are shown with crossing line representing mean value).C IF analysis for selected markers in mouse prostate tissue and organoid sections (scale bar = 50 μm).D Representative stereoscopic images of mouse prostate organoids cultured in the absence of A83-01, with or without Follistatin (Fst, 500 ng/mL, 6 days), or with or without the Activin A neutralizing antibody MAB3381 (1 μg/mL, 6 days). Scale bar = 1 mmE Western blot (WB) analysis in mouse prostate organoids for selected canonical and non-canonical Tgf-β signalling mediators, and the cell cycle inhibitor p21 (Activin A, 50 ng/mL; Tgfb1, 500 ng/mL; 24 hours).G Representative stereoscopic images of mouse prostate organoids following treatment with Takinib (Tak1 inhibitor; 5 μM, 6 days), SB203580 (p38α/β inhibitor; 10 μM, 6 days) or the structurally unrelated Ralimetinib (p38α/β inhibitor; 1 μM, 7 days). Scale bar = 1 mmH Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected signalling mediators in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 hours).I Gene set enrichment analysis (GSEA) plots displaying significant enrichment for inflammatory response and Tumor Necrosis Factor (TNF) signalling in mouse prostate organoids cultured without A83-01 (24 hours) versus complete medium (ENRAD).J Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected cell cycle regulators, in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 or 48 hours).


Figure 3A Diagram depicting the expansion of mouse prostate organoid cultures in the absence of the Tgf - β ligand inhibitor A83 - 01 ( n = 3 biological replicates per lobe - specific organoid culture ; arrow = continuous expansion ; dot = passage ; cross = culture loss ) . B Representative stereoscopic images of C57BL / 6J DLP # 1 and # 3 mouse prostate organoid lines during adaptation in the absence of A83 - 01 . Scale bar = 1 mmD Gene set enrichment analysis ( GSEA ) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines ( C57 # 1 DLP , top ; C57 # 3 DLP , bottom ) adapted to grow in the absence of A83 - 01 vs . normal control organoids cultured in complete medium ( ENRAD ) . E Western blot analysis in C57 # 1 and C57 # 3 DLP mouse prostate organoid lines adapted to grow in the absence of A83 - 01 vs . normal control organoids cultured in complete medium ( ENRAD ) . Immunoblots are displayed for Chek1 ( ATR signalling mediator ) , Trp53 and p21 . F Representative spectral karyotype ( SKY ) images of metaphase spreads obtained from C57 # 1 DLP mouse prostate organoids cultured in normal conditions ( ENRAD ) or upon adaptation without A83 - 01 . Widespread genomic instability is observed following adaptation . Scale bars = 5 µm . G - H Representative telomere FISH images - and quantification ( n = 2 biological replicates , 20 metaphases per condition ) - in C57 # 1 DLP mouse prostate organoids cultured in normal conditions ( ENRAD ) or upon adaptation without A83 - 01 . Widespread telomeric instability is observed following adaptation ( arrows indicate telomere doublets , One - way ANOVA , scale bars = 10 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 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 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 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

Figure 3A Diagram depicting the expansion of mouse prostate organoid cultures in the absence of the Tgf-β ligand inhibitor A83-01 (n = 3 biological replicates per lobe-specific organoid culture; arrow = continuous expansion; dot = passage; cross = culture loss).B Representative stereoscopic images of C57BL/6J DLP #1 and #3 mouse prostate organoid lines during adaptation in the absence of A83-01. Scale bar = 1 mmD Gene set enrichment analysis (GSEA) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines (C57#1 DLP, top; C57#3 DLP, bottom) adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD).E Western blot analysis in C57#1 and C57#3 DLP mouse prostate organoid lines adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD). Immunoblots are displayed for Chek1 (ATR signalling mediator), Trp53 and p21.F Representative spectral karyotype (SKY) images of metaphase spreads obtained from C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread genomic instability is observed following adaptation. Scale bars = 5 µm. G-H Representative telomere FISH images - and quantification (n = 2 biological replicates, 20 metaphases per condition) - in C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread telomeric instability is observed following adaptation (arrows indicate telomere doublets, One-way ANOVA, scale bars = 10 µm).


Figure 1A - Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C . trachomatis L2 ( multiplicity of infection MOI = 1 ) in the presence or not of BP . In the indicated samples CP4d was added 2 h before infection . Cell lysates were run on SDS - PAGE , proteins were transferred to a membrane and BP incorporation was revealed with HRP - conjugated streptavidin . BP incorporation is enhanced in infected samples , and is inhibited by CP4d . The two main bands present in all samples correspond to naturally biotinylated host proteins ( Haneji & Koide , 1989 ) . After blotting the membrane was stained with Coomassie blue to control for equal loading . B - Same as in ( A ) , except that where indicated 250 μM doxycycline ( doxy , left ) or 7 μM cycloheximide ( CHX , right ) were added 24 h or 2 hpi , respectively . C - Whole cell lysates were prepared with TG2 + / + and TG2 - / - MEFs infected or not for 48 h with C . trachomatis L2 in the presence or not of BP , and analyzed as in ( A ) . D - Western blot with anti - TG2 antibodies on total cell lysates infected or not with C . trachomatis L2 for the indicated time . The histogram displays the mean ± SD of TG2 expression relative to actin from four independent experiments , with the results of the Student ' s ratio - paired t - test . NI : not infected . E - Cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 46 h . Where indicated , 40 μM CP4d was added 2 hpi . tgm2 transcripts were measured by real - time RT - PCR and normalized to actin transcripts following the ∆ ∆ Ct method . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least four times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown . F - Cells were treated for 18 h with the indicated concentration of human recombinant IL - 6 before measuring TG2 transcription relative to actin like in ( E ) . The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least three times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown . Cells were left uninfected or infected with C . trachomatis L2 in the presence of the indicated concentration of anti - IL - 6 receptor antibodies . Forty - eight h later TG2 transcription relative to actin was measured like in ( E ) . The data are presented as relative mRNA levels compared to uninfected / untreated cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least three times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

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

Figure 1A - Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C. trachomatis L2 (multiplicity of infection MOI=1) in the presence or not of BP. In the indicated samples CP4d was added 2 h before infection. Cell lysates were run on SDS-PAGE, proteins were transferred to a membrane and BP incorporation was revealed with HRP-conjugated streptavidin. BP incorporation is enhanced in infected samples, and is inhibited by CP4d. The two main bands present in all samples correspond to naturally biotinylated host proteins (Haneji & Koide, 1989). After blotting the membrane was stained with Coomassie blue to control for equal loading. B - Same as in (A), except that where indicated 250 μM doxycycline (doxy, left) or 7 μM cycloheximide (CHX, right) were added 24 h or 2 hpi, respectively.C - Whole cell lysates were prepared with TG2+/+ and TG2-/- MEFs infected or not for 48 h with C. trachomatis L2 in the presence or not of BP, and analyzed as in (A).D -Western blot with anti-TG2 antibodies on total cell lysates infected or not with C. trachomatis L2 for the indicated time. The histogram displays the mean ± SD of TG2 expression relative to actin from four independent experiments, with the results of the Student's ratio-paired t-test. NI: not infected.E - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 46 h. Where indicated, 40 μM CP4d was added 2 hpi. tgm2 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least four times. P-values of Student's ratio-paired t-test <0.05 are shown.F - Cells were treated for 18 h with the indicated concentration of human recombinant IL-6 before measuring TG2 transcription relative to actin like in (E). The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least three times. P-values of Student's ratio-paired t-test <0.05 are shown.Cells were left uninfected or infected with C. trachomatis L2 in the presence of the indicated concentration of anti-IL-6 receptor antibodies. Forty-eight h later TG2 transcription relative to actin was measured like in (E). The data are presented as relative mRNA levels compared to uninfected/untreated cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least three times. P-values of Student's ratio-paired t-test <0.05 are shown.


Figure 2A - HeLa cells were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with L2incDGFP at MOI = 0 . 15 . Thirty hours later the cells were disrupted and bacterial titers ( IFU = inclusion forming unit ) were determined by re - infecting fresh HeLa cells as described in the methods . The mean ± SD of three independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 . B - HeLa cells were transfected with control siRNA or two siRNAs against TG2 . Two days later , the efficiency of the silencing was assessed by western blot using anti - TG2 antibodies and anti - actin antibodies as loading control ( bottom ) . Duplicate wells were infected with L2incDGFP and progeny was analyzed as in ( A ) ( top ) . The mean ± SD of four independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 . C - Same as in ( A ) except that C . trachomatis serovar L2 ( left ) or D ( right ) were grown in primary cells isolated from fallopian tubes . For serovar D , IFU were determined 48 hpi . The mean ± SD of four to five independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 . D - HeLa cells were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with L2incDGFP at MOI = 0 . 15 . Thirty hours later the cells were fixed and analyzed by flow cytometry . The percentage of infected cells ( left ) and the mean fluorescence of the infected population ( right ) ± SD are shown for three independent experiments . P - values of Student ' s paired t - test are indicated when < 0 . 05 . A representative field for each condition is shown , scale bar = 10 μm . E - Primary epithelial cells isolated from fallopian tubes were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with C . trachomatis serovar L2 ( left ) or D ( right ) . Twenty - four hours later the cells were fixed , bacteria were stained using FITC - labeled anti - Chlamydia - LPS antibodies , and the mean size of the inclusions manually determined using ImageJ , on twenty inclusions per experiment . The mean ± SD of three independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 . F - Mice were infected intravaginally with 105 IFU of C . muridarum . Twenty - five days later the mice were sacrificed and the upper genital tract , from the uterine horn to the oviduct , was collected . The right part was used for bacterial burden assessment ( top left ) . The left part was rinsed with PBS and observed with a binocular magnifier ( right ) to determine the hydrosalpinx score ( bottom left ) . Each dot represents one mouse , the mean ± SD is shown . P - values of Mann - Whitney test are indicated when < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were disrupted and bacterial titers (IFU=inclusion forming unit) were determined by re-infecting fresh HeLa cells as described in the methods. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.B - HeLa cells were transfected with control siRNA or two siRNAs against TG2. Two days later, the efficiency of the silencing was assessed by western blot using anti-TG2 antibodies and anti-actin antibodies as loading control (bottom). Duplicate wells were infected with L2incDGFP and progeny was analyzed as in (A) (top). The mean ± SD of four independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.C - Same as in (A) except that C. trachomatis serovar L2 (left) or D (right) were grown in primary cells isolated from fallopian tubes. For serovar D, IFU were determined 48 hpi. The mean ± SD of four to five independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.D - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were fixed and analyzed by flow cytometry. The percentage of infected cells (left) and the mean fluorescence of the infected population (right) ± SD are shown for three independent experiments. P-values of Student's paired t-test are indicated when <0.05. A representative field for each condition is shown, scale bar = 10 μm.E - Primary epithelial cells isolated from fallopian tubes were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with C. trachomatis serovar L2 (left) or D (right). Twenty-four hours later the cells were fixed, bacteria were stained using FITC-labeled anti-Chlamydia-LPS antibodies, and the mean size of the inclusions manually determined using ImageJ, on twenty inclusions per experiment. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.F - Mice were infected intravaginally with 105 IFU of C. muridarum. Twenty-five days later the mice were sacrificed and the upper genital tract, from the uterine horn to the oviduct, was collected. The right part was used for bacterial burden assessment (top left). The left part was rinsed with PBS and observed with a binocular magnifier (right) to determine the hydrosalpinx score (bottom left). Each dot represents one mouse, the mean ± SD is shown. P-values of Mann-Whitney test are indicated when <0.05.


Figure 3A - MEFs were grown for 24 h culture medium complemented with the indicated concentration of glucose before being infected with L2 IncDGFP bacteria ( MOI = 0 . 2 ) . Cells were disrupted 30 h later and the bacterial titer determined by re - infecting fresh wild type cells . The mean ± SD of three independent experiments are shown . B - Cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 48 h . Where indicated 40 μM CP4d was added 2 hpi . GLUT - 1 and GLUT - 3 transcripts were measured by real - time RT - PCR and normalized to actin transcripts following the ∆ ∆ Ct method . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated four times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 . C - Relationship between TGM2 and GLUT - 1 ( top ) and GLUT - 3 ( bottom ) expression across 265 HGSOCs . Expression comparisons were performed using Spearman ' s rank correlation test . D - HeLa cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 46 h . HIF - 1α transcripts were measured by real - time RT - PCR and normalized to actin transcripts . The data are presented as relative E - HeLa cells were transfected with control siRNA or two siRNAs against HIF - 1α . Two days later , cells were infected with C . trachomatis L2 ( MOI = 1 ) for 48 h . The indicated transcripts were measured by real - time RT - PCR and normalized to actin transcripts . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated three times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 . F - TG2 - / - MEFs stably transformed or not with the indicated TG2 construct were infected for two days with C . trachomatis L2 ( MOI = 1 ) . Mouse GLUT - 1 transcripts were measured by real - time RT - PCR and normalized to mouse actin transcripts . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated three times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

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

Figure 3A - MEFs were grown for 24 h culture medium complemented with the indicated concentration of glucose before being infected with L2 IncDGFP bacteria (MOI = 0.2). Cells were disrupted 30 h later and the bacterial titer determined by re-infecting fresh wild type cells. The mean ± SD of three independent experiments are shown.B - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 48 h. Where indicated 40 μM CP4d was added 2 hpi. GLUT-1 and GLUT-3 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated four times. P-values of Student's ratio-paired t-test are indicated when <0.05.C -Relationship between TGM2 and GLUT-1 (top) and GLUT-3 (bottom) expression across 265 HGSOCs. Expression comparisons were performed using Spearman's rank correlation test.D -HeLa cells were infected with C. trachomatis L2 (MOI=1) for 24 or 46 h. HIF-1α transcripts were measured by real-time RT-PCR and normalized to actin transcripts. The data are presented as relative E - HeLa cells were transfected with control siRNA or two siRNAs against HIF-1α. Two days later, cells were infected with C. trachomatis L2 (MOI=1) for 48 h. The indicated transcripts were measured by real-time RT-PCR and normalized to actin transcripts. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated three times. P-values of Student's ratio-paired t-test are indicated when <0.05.F - TG2-/- MEFs stably transformed or not with the indicated TG2 construct were infected for two days with C. trachomatis L2 (MOI=1). Mouse GLUT-1 transcripts were measured by real-time RT-PCR and normalized to mouse actin transcripts. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated three times. P-values of Student's ratio-paired t-test are indicated when <0.05.


Figure 4A - HeLa cells were infected with C . trachomatis ( MOI = 1 ) and 40 µM CP4d was added or not 2 hpi . After 24 h 0 . 5 mM BP was added and cells were lysed at 48 hpi . Lysates were precipitated with streptavidin - coated beads . After separation with SDS - PAGE , proteins were transferred to a membrane and blotted with anti - GFPT antibody followed with HRP - conjugated secondary antibody . B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP . Samples were incubated for 3 h at 37 ° C before separation by SDS - PAGE . Proteins were transferred to a membrane and BP was revealed using HRP - conjugated streptavidin . rhGFPT1 is 77 . 5 kDa . D - In vitro assay was performed as described in B using wild type rhGFPT1 ( WT ) , rhGFPT1 Q58N , rhGFPT1 Q328N or rhGFPT1 Q555N as substrates . The reaction was performed at 37 ° C for 30 min . After probing with HRP - Streptavidin the membrane was washed and probed with anti - GFPT antibodies followed with HRP - conjugated secondary antibodies . The ratio of modified protein ( streptavidin signal ) to the total GFPT is shown , normalized to its value with WT rhGFPT1 . The mean ± SD of five independent experiments is shown , the p - value of the Student ' s ratio - paired t - test is indicated when < 0 . 05 . E - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 ° C for 45 min with fructose - 6 - P and glutamine . The production of glucosamine - 6 - P was measured using HPAEC - PAD . Results of three independent experiments are shown , with mean ± SD , and p - value of the Student ' s paired t - test is indicated ( * P < 0 . 05 ) .

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

Figure 4A - HeLa cells were infected with C. trachomatis (MOI = 1) and 40 µM CP4d was added or not 2 hpi. After 24 h 0.5 mM BP was added and cells were lysed at 48 hpi. Lysates were precipitated with streptavidin-coated beads. After separation with SDS-PAGE, proteins were transferred to a membrane and blotted with anti-GFPT antibody followed with HRP-conjugated secondary antibody.B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP. Samples were incubated for 3 h at 37°C before separation by SDS-PAGE. Proteins were transferred to a membrane and BP was revealed using HRP-conjugated streptavidin. rhGFPT1 is 77.5 kDa.D - In vitro assay was performed as described in B using wild type rhGFPT1 (WT), rhGFPT1 Q58N, rhGFPT1 Q328N or rhGFPT1 Q555N as substrates. The reaction was performed at 37 °C for 30 min. After probing with HRP-Streptavidin the membrane was washed and probed with anti-GFPT antibodies followed with HRP-conjugated secondary antibodies. The ratio of modified protein (streptavidin signal) to the total GFPT is shown, normalized to its value with WT rhGFPT1. The mean ± SD of five independent experiments is shown, the p-value of the Student's ratio-paired t-test is indicated when <0.05.E - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 °C for 45 min with fructose-6-P and glutamine. The production of glucosamine-6-P was measured using HPAEC-PAD. Results of three independent experiments are shown, with mean ± SD, and p-value of the Student's paired t-test is indicated (*P < 0.05).


Figure 5B - Endocervical epithelial cells were pre - treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin ( or an equivalent volume of DMSO ) and 0 . 5 µM BP . Six hours later whole cell lysates were analyzed by western blot . The membrane was first blotted with HRP - conjugated streptavidin to detect TG2 activity , then extensively washed and probed with anti - O - GlcNAcylation antibody followed with HRP - conjugated secondary antibodies . Last the membrane was probed with anti - actin as a loading control . C - The same experimental procedure as described in ( B ) was applied to HeLa cells treated for 48 h prior to ionomycin treatment ( 8 µM ) with siRNA control or directed against TG2 .

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

Figure 5B - Endocervical epithelial cells were pre-treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin (or an equivalent volume of DMSO) and 0.5 µM BP. Six hours later whole cell lysates were analyzed by western blot. The membrane was first blotted with HRP-conjugated streptavidin to detect TG2 activity, then extensively washed and probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. Last the membrane was probed with anti-actin as a loading control.C - The same experimental procedure as described in (B) was applied to HeLa cells treated for 48 h prior to ionomycin treatment (8 µM) with siRNA control or directed against TG2.


Figure 6A - HeLa cells were infected or not ( NI ) with C . trachomatis ( MOI = 1 ) , then lysed 24 or 48 hpi . After separation with SDS - PAGE , proteins were transferred to a membrane , probed with anti - O - GlcNAcylation antibody followed with HRP - conjugated secondary antibodies . After extensive washes the membrane was blotted again with anti - GFPT and anti - actin antibodies before revelation with HRP - conjugated secondary antibodies . B - HeLa cells were infected or not with C . trachomatis ( MOI = 1 ) . Twenty - four hours later , 8 µM ionomycin ( or DMSO alone ) and 0 . 5 mM BP were added . After 6 h of treatment , cells were lysed and proteins revealed as in ( A ) . C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed . After separation with SDS - PAGE , proteins were transferred to a membrane , probed with anti - GFPT and anti - actin antibody before revelation with HRP - conjugated secondary antibodies . D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2 . Two days later the cells were infected for the indicated times ( MOI = 0 . 3 ) before fixation , rupture of the cells and measurement of bacterial diameter . The mean diameter ± SD and p - values of Student ' s paired t - test on 4 independent experiments are shown . E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not ( siCTRL ) were infected with C . trachomatis ( MOI = 0 . 15 ) . Thirty hours later cells were fixed and analyzed by flow cytometry . The percentage of infected cells ( top ) and the mean fluorescence of the infected population ( middle ) ) ± SD are shown for at least four independent experiments . Duplicate wells were lysed and used to re - infect fresh HeLa cells to determine the bacterial titer ( bottom ) . P - values of Student ' s ratio - paired t - test are indicated .

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

Figure 6A - HeLa cells were infected or not (NI) with C. trachomatis (MOI = 1), then lysed 24 or 48 hpi. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. After extensive washes the membrane was blotted again with anti-GFPT and anti-actin antibodies before revelation with HRP-conjugated secondary antibodies.B - HeLa cells were infected or not with C. trachomatis (MOI = 1). Twenty-four hours later, 8 µM ionomycin (or DMSO alone) and 0.5 mM BP were added. After 6 h of treatment, cells were lysed and proteins revealed as in (A).C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-GFPT and anti-actin antibody before revelation with HRP-conjugated secondary antibodies.D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2. Two days later the cells were infected for the indicated times (MOI=0.3) before fixation, rupture of the cells and measurement of bacterial diameter. The mean diameter ± SD and p-values of Student's paired t-test on 4 independent experiments are shown.E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not (siCTRL) were infected with C. trachomatis (MOI = 0.15). Thirty hours later cells were fixed and analyzed by flow cytometry. The percentage of infected cells (top) and the mean fluorescence of the infected population (middle) ) ± SD are shown for at least four independent experiments. Duplicate wells were lysed and used to re-infect fresh HeLa cells to determine the bacterial titer (bottom). P-values of Student's ratio-paired t-test are indicated.


Figure 1IFN - β promoter activity ( A ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected HEK293T cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . ISRE reporter activity ( B ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected HEK293T cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . IFN - β promoter activity ( C ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected H1299 cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . ISRE reporter activity ( D ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected H1299 cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . IFN - β promoter activity ( E ) in the indicated siRNA - transfected H1299 cells ( si - NC , si - SIRT5 # 1 and si - SIRT5 # 2 ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . NC , negative control . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . ISRE reporter activity ( F ) in the indicated siRNA - transfected H1299 cells ( si - NC , si - SIRT5 # 1 and si - SIRT5 # 2 ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . NC , negative control . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . IFN - β promoter activity ( G ) in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) or the wildtype ( WT ) H1299 cells ( SIRT5 + / + ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . ISRE reporter activity ( H ) in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) or the wildtype ( WT ) H1299 cells ( SIRT5 + / + ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . I ISRE reporter activity in Myc - SIRT5 ( 0 , 100 ng or 200 ng ) - transfected HEK293T cells with or without poly ( I : C ) transfection for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . J ISRE reporter activity in co - transfection of Myc - RIG - I ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200ng ) - transfected HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . K ISRE reporter activity in co - transfection of Myc - MDA5 ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . L ISRE reporter activity in co - transfection of HA - MAVS ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . M ISRE reporter activity in co - transfection of Myc - TBK1 ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . N ISRE reporter activity in co - transfection of HA - IRF3 ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . O ISRE reporter activity in co - transfection of HA - MAVS ( 200 ng ) together with Myc - SIRT5 ( 200ng ) or Myc - SIRT5 - H158Y ( 200ng ) respectively in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s 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 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 O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1IFN-β promoter activity (A) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected HEK293T cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).ISRE reporter activity (B) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected HEK293T cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).IFN-β promoter activity (C) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected H1299 cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).ISRE reporter activity (D) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected H1299 cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).IFN-β promoter activity (E) in the indicated siRNA-transfected H1299 cells (si-NC, si-SIRT5#1 and si-SIRT5#2) with or without SeV infection (SeV or UI) for 18~24 h. NC, negative control. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).ISRE reporter activity (F) in the indicated siRNA-transfected H1299 cells (si-NC, si-SIRT5#1 and si-SIRT5#2) with or without SeV infection (SeV or UI) for 18~24 h. NC, negative control. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).IFN-β promoter activity (G) in SIRT5-deficient H1299 cells (SIRT5 -/-) or the wildtype (WT) H1299 cells (SIRT5 +/+) with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).ISRE reporter activity (H) in SIRT5-deficient H1299 cells (SIRT5 -/-) or the wildtype (WT) H1299 cells (SIRT5 +/+) with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).I ISRE reporter activity in Myc-SIRT5 (0, 100 ng or 200 ng)-transfected HEK293T cells with or without poly(I:C) transfection for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).J ISRE reporter activity in co-transfection of Myc-RIG-I (200 ng) together with Myc-SIRT5 (0, 100 ng or 200ng)-transfected HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).K ISRE reporter activity in co-transfection of Myc-MDA5 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).L ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).M ISRE reporter activity in co-transfection of Myc-TBK1 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).N ISRE reporter activity in co-transfection of HA-IRF3 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).O ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (200ng) or Myc-SIRT5-H158Y(200ng) respectively in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


Figure 2A Confocal microscopy image of endogenous SIRT5 co - localized with endogenous MAVS in H1299 cells detected by immunofluorescence staining using anti - SIRT5 and anti - MAVS antibodies . Mito , Mitotracker ; Scale bar = 8 µm . B - C Co - immunoprecipitation of Myc - SIRT5 with HA - MAVS and vice versa . HEK293T cells were co - transfected with indicated plasmids for 24 h . Anti - Myc ( B ) or anti - HA antibody conjugated agarose beads ( C ) were used for immunoprecipitation and the interaction was detected by immunoblotting with the indicated antibodies . Data information : IP , immunoprecipitation ; TCL , total cell lysates . D Endogenous interaction between MAVS and SIRT5 . Anti - MAVS antibody was used for immunoprecipitation , and normal mouse IgG was used as a control . Data information : IP , immunoprecipitation ; TCL , total cell lysates . E Endogenous interaction between MAVS and SIRT5 in the wildtype ( WT ) ( MAVS + / + ) or SIRT5 - deficient ( MAVS - / - ) H1299 cells . Anti - MAVS antibody was used for immunoprecipitation , and the interaction was detected by immunoblotting with anti - SIRT5 antibody . Data information : IP , immunoprecipitation ; TCL , total cell lysates . F GST pull - down assay for GST - tagged MAVS and His - tagged SIRT5 . GST - tagged MAVS and His - tagged SIRT5 were expressed in Escherichia coli ( BL21 ) , respectively . The association of GST - MAVS with His - SIRT5 was detected by immunoblotting with anti - SIRT5 antibody . GST and GST - MAVS proteins were stained with Coomassie blue . H - I Co - immunoprecipitation analysis of HA - SIRT5 with Flag - MAVS - truncated mutants . HEK293T cells were co - transfected with the indicated plasmids . Anti - Flag antibody conjugated agarose beads were used for immunoprecipitation and the interaction was analyzed by immunoblotting with the indicated antibodies . Flag - MAVS fragments ( WT : full length ; ΔC1 , 1 - 173 aa ; ΔC2 , 1 - 513 aa ; ΔN1 , 91 - 540 aa ; ΔN2 , 173 - 540 aa ; ΔCARD , Δ10 - 77 aa ; ΔPR , Δ103 - 173 aa ; ΔTM , Δ514 - 535 aa ) . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O 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 Confocal microscopy image of endogenous SIRT5 co-localized with endogenous MAVS in H1299 cells detected by immunofluorescence staining using anti-SIRT5 and anti-MAVS antibodies. Mito, Mitotracker; Scale bar = 8 µm.B-C Co-immunoprecipitation of Myc-SIRT5 with HA-MAVS and vice versa. HEK293T cells were co-transfected with indicated plasmids for 24 h. Anti-Myc (B) or anti-HA antibody conjugated agarose beads (C) were used for immunoprecipitation and the interaction was detected by immunoblotting with the indicated antibodies. Data information: IP, immunoprecipitation; TCL, total cell lysates.D Endogenous interaction between MAVS and SIRT5. Anti-MAVS antibody was used for immunoprecipitation, and normal mouse IgG was used as a control. Data information: IP, immunoprecipitation; TCL, total cell lysates.E Endogenous interaction between MAVS and SIRT5 in the wildtype (WT) (MAVS +/+) or SIRT5-deficient (MAVS -/-) H1299 cells. Anti-MAVS antibody was used for immunoprecipitation, and the interaction was detected by immunoblotting with anti-SIRT5 antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.F GST pull-down assay for GST-tagged MAVS and His-tagged SIRT5. GST-tagged MAVS and His-tagged SIRT5 were expressed in Escherichia coli (BL21), respectively. The association of GST-MAVS with His-SIRT5 was detected by immunoblotting with anti-SIRT5 antibody. GST and GST-MAVS proteins were stained with Coomassie blue.H-I Co-immunoprecipitation analysis of HA-SIRT5 with Flag-MAVS-truncated mutants. HEK293T cells were co-transfected with the indicated plasmids. Anti-Flag antibody conjugated agarose beads were used for immunoprecipitation and the interaction was analyzed by immunoblotting with the indicated antibodies. Flag-MAVS fragments (WT: full length; ΔC1, 1-173 aa; ΔC2, 1-513 aa; ΔN1, 91-540 aa; ΔN2, 173-540 aa; ΔCARD, Δ10-77 aa; ΔPR, Δ103-173 aa; ΔTM, Δ514-535 aa). Data information: IP, immunoprecipitation; TCL, total cell lysates.


Figure 3A - B MAVS could be succinylated in vitro . GST - MAVS ( A ) extracted from E . coli or Flag - tagged MAVS ( B ) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl - CoA . Protein succinylation was detected with anti - pan - succinyl - lysine antibody ; GST - MAVS or Flag - MAVS was stained by Coomassie blue . C The succinylated residue in MAVS was identified by mass spectrometry analysis . E Dot - blot assay for the specificity of anti - succ - K7 - MAVS antibody . Equal amounts of succinyl - peptides or the control peptides were immunoblotted with the indicated dilutions of anti - succ - K7 - MAVS antibody . GST - MAVS ( F ) extracted from E . coli cells was incubated with the indicated concentrations of succinyl - CoA . The succinylation of MAVS at lysine 7 was detected by anti - succ - K7 - MAVS antibody . Flag - MAVS ( G ) purified from HEK293T cells was incubated with the indicated concentrations of succinyl - CoA . The succinylation of MAVS at lysine 7 was detected by anti - succ - K7 - MAVS antibody . H Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5 - intact H1299 cells ( SIRT5 + / + ) ( 3 . 2 vs . 1 . 0 ) . The cell lysates from SIRT5 - deficient H1299 cells or the SIRT5 - intact H1299 cells were immunoprecipitated with anti - MAVS antibody or mouse IgG control , followed by immunoblotting with anti - succ - K7 - MAVS antibody . Data information : IP , immunoprecipitation ; TCL , total cell lysates . I Reconstitution of WT SIRT5 in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) caused a significant reduction in succinylation of MAVS at Lys 7 ( 1 . 0 vs . 0 . 2 ) ; but overexpression of SIRT5 - H158Y in SIRT5 - / - H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 ( 1 . 0 vs . 1 . 2 ) . The SIRT5 - / - H1299 cells were transfected with Flag - SIRT5 or Flag - SIRT5 - H158Y , followed by immunoprecipitating with anti - MAVS antibody or mouse IgG control , and immunoblotting with anti - succ - K7 - MAVS antibody . Data information : IP , immunoprecipitation ; TCL , total cell lysates . J - K Knockout of Sirt5 increased Mavs succinylation in mouse livers ( J ) and lungs ( K ) . Proteins extracted from livers ( J ) and lungs ( K ) of Sirt5 KO and the wildtype littermates ( n = 3 per group ) were detected by the indicated antibodies . MAVS succinylation was determined by anti - succ - K7 - MAVS antibody , and anti - pan - succinyl - lysine antibody was used as positive controls .

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

Figure 3A-B MAVS could be succinylated in vitro. GST-MAVS (A) extracted from E.coli or Flag-tagged MAVS (B) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. Protein succinylation was detected with anti-pan-succinyl-lysine antibody; GST-MAVS or Flag-MAVS was stained by Coomassie blue.C The succinylated residue in MAVS was identified by mass spectrometry analysis.E Dot-blot assay for the specificity of anti-succ-K7-MAVS antibody. Equal amounts of succinyl-peptides or the control peptides were immunoblotted with the indicated dilutions of anti-succ-K7-MAVS antibody.GST-MAVS (F) extracted from E.coli cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody.Flag-MAVS (G) purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody.H Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5-intact H1299 cells (SIRT5 +/+) (3.2 vs. 1.0). The cell lysates from SIRT5-deficient H1299 cells or the SIRT5-intact H1299 cells were immunoprecipitated with anti-MAVS antibody or mouse IgG control, followed by immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.I Reconstitution of WT SIRT5 in SIRT5-deficient H1299 cells (SIRT5 -/-) caused a significant reduction in succinylation of MAVS at Lys 7 (1.0 vs. 0.2); but overexpression of SIRT5-H158Y in SIRT5 -/- H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 (1.0 vs. 1.2). The SIRT5 -/- H1299 cells were transfected with Flag-SIRT5 or Flag-SIRT5-H158Y, followed by immunoprecipitating with anti-MAVS antibody or mouse IgG control, and immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.J-K Knockout of Sirt5 increased Mavs succinylation in mouse livers (J) and lungs (K). Proteins extracted from livers (J) and lungs (K) of Sirt5 KO and the wildtype littermates (n = 3 per group) were detected by the indicated antibodies. MAVS succinylation was determined by anti-succ-K7-MAVS antibody, and anti-pan-succinyl-lysine antibody was used as positive controls.


Figure 4A - C Quantitative real time PCR analysis ( qPCR ) of IFNβ ( A ) , CXCL10 ( B ) and IFIT1 ( C ) mRNA in HEK293T cells transfected with the control plasmid ( Myc empty ) or the plasmid expressing Myc - SIRT5 ( Myc - SIRT5 ) for 24 h , followed by infection with or without SeV ( SeV or UI ) for 8h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . D - F qPCR analysis of IFNβ ( D ) , CXCL10 ( E ) , and IFIT1 ( F ) mRNA in SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) infected with or without SeV ( SeV or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . G - I qPCR analysis of IFNβ ( G ) , CXCL10 ( H ) and IFIT1 ( I ) mRNA in SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) infected with or without VSV ( VSV or UI ) for 8h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . J qPCR analysis of IFNβ mRNA in HEK293T cells transfected with the control plasmid ( Myc empty ) , or the plasmid expressing Myc - SIRT5 ( Myc - SIRT5 ) , or its enzyme - deficient mutant H158Y ( Myc - SIRT5 - H158Y ) for 24 h , followed by stimulation with or without poly ( I : C ) for 8 h . Data information : All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . K qPCR analysis of IFNβ mRNA in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) transfected with the control plasmid ( Myc empty ) or the plasmid expressing Myc - SIRT5 ( Myc - SIRT5 ) or its enzyme - deficient mutant H158Y ( Myc - SIRT5 - H158Y ) for 24 h , followed by infection with ( + ) or without ( - ) SeV for 8 h . Data information : All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . L SDD - AGE analysis of MAVS aggregates in HEK293T cells transfected HA - MAVS together with WT Myc - SIRT5 or its enzyme - deficient mutant ( Myc - SIRT5 - H158Y ) after infected with SeV for 12h . SDS - PAGE immunoblotting was used as a loading control . M - N IFN - β promoter activity ( M ) and ISRE reporter activity ( N ) in HEK293T cells co - transfected Myc - SIRT5 together with HA - MAVS or HA - MAVS - K7R . Data information All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) were infected with VSV - GFP viruses ( MOI = 0 . 1 ) for 12 h , and viral infectivity was detected by fluorescence microscopy ( O ) SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) were infected with VSV - GFP viruses ( MOI = 0 . 1 ) for 12 h and viral infectivity western blot analysis ( P ) Data information : ll data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) were infected with VSV - GFP viruses ( MOI = 0 . 1 ) for 12 h , and viral infectivity was detected by flow cytometry analysis ( n = 3 ) ( Q ) .

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

Figure 4A-C Quantitative real time PCR analysis (qPCR) of IFNβ (A), CXCL10 (B) and IFIT1 (C) mRNA in HEK293T cells transfected with the control plasmid (Myc empty) or the plasmid expressing Myc-SIRT5 (Myc-SIRT5) for 24 h, followed by infection with or without SeV (SeV or UI) for 8h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).D-F qPCR analysis of IFNβ (D), CXCL10 (E), and IFIT1 (F) mRNA in SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) infected with or without SeV (SeV or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).G-I qPCR analysis of IFNβ (G), CXCL10 (H) and IFIT1 (I) mRNA in SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) infected with or without VSV (VSV or UI) for 8h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).J qPCR analysis of IFNβ mRNA in HEK293T cells transfected with the control plasmid (Myc empty), or the plasmid expressing Myc-SIRT5 (Myc-SIRT5), or its enzyme-deficient mutant H158Y (Myc-SIRT5-H158Y) for 24 h, followed by stimulation with or without poly(I:C) for 8 h. Data information: All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).K qPCR analysis of IFNβ mRNA in SIRT5-deficient H1299 cells (SIRT5-/-) transfected with the control plasmid (Myc empty) or the plasmid expressing Myc-SIRT5 (Myc-SIRT5) or its enzyme-deficient mutant H158Y (Myc-SIRT5-H158Y) for 24 h, followed by infection with (+) or without (-) SeV for 8 h. Data information: All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).L SDD-AGE analysis of MAVS aggregates in HEK293T cells transfected HA-MAVS together with WT Myc-SIRT5 or its enzyme-deficient mutant (Myc-SIRT5-H158Y) after infected with SeV for 12h. SDS-PAGE immunoblotting was used as a loading control.M-N IFN-β promoter activity (M) and ISRE reporter activity (N) in HEK293T cells co-transfected Myc-SIRT5 together with HA-MAVS or HA-MAVS-K7R. Data information All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) were infected with VSV-GFP viruses (MOI = 0.1) for 12 h, and viral infectivity was detected by fluorescence microscopy (O)SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) were infected with VSV-GFP viruses (MOI = 0.1) for 12 h and viral infectivity western blot analysis (P) Data information: ll data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) were infected with VSV-GFP viruses (MOI = 0.1) for 12 h, and viral infectivity was detected by flow cytometry analysis (n = 3) (Q).


Figure 5A - F qPCR analysis of Ifnβ ( A ) , Ifnα1 ( B ) , Ifnα4 ( C ) , Ifit1 ( D ) , Cxcl10 ( E ) , and Cxcl11 ( F ) mRNA in WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without SeV ( SeV or UI ) for 8h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . G WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without SeV for the indicated times , and the cell lysates were analyzed by immunoblotting for monomeric ( Monomer ) and dimeric ( Dimer ) Irf3 ( top ; native - PAGE ) , phosphorylated Irf3 ( p - Irf3 ) , total Irf3 , Sirt5 , and GAPDH ( bottom ) . H WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without SeV for 8 h , the interaction between endogenous Mavs and Rig - I was revealed by immunoprecipitation using anti - Mavs antibody , followed by immunoblotting with anti - Rig - I antibody . I SDD - AGE analysis of Mavs aggregates in WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) infected with SeV for 12 h . SDS - PAGE immunoblotting was used as a loading control . J - O qPCR analysis of Ifnβ ( J ) , Ifnα1 ( K ) , Ifnα4 ( L ) , Ifit1 ( M ) , Cxcl10 ( N ) , and Cxcl11 ( O ) mRNA in WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without VSV ( VSV or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) . WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without VSV - GFP viruses for 12 h , and viral infectivity was detected by fluorescence microscopy ( P ) WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without VSV - GFP viruses for 12 h , and viral infectivity was detected by western blot analysis ( Q ) WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without VSV - GFP viruses for 12 h , and viral infectivity was detected by flow cytometry analysis ( n = 3 ) ( R ) . Data information : All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 5A-F qPCR analysis of Ifnβ (A), Ifnα1 (B), Ifnα4 (C), Ifit1 (D), Cxcl10 (E), and Cxcl11 (F) mRNA in WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) infected with or without SeV (SeV or UI) for 8h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).G WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without SeV for the indicated times, and the cell lysates were analyzed by immunoblotting for monomeric (Monomer) and dimeric (Dimer) Irf3 (top; native-PAGE), phosphorylated Irf3 (p-Irf3), total Irf3, Sirt5, and GAPDH (bottom).H WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without SeV for 8 h, the interaction between endogenous Mavs and Rig-I was revealed by immunoprecipitation using anti-Mavs antibody, followed by immunoblotting with anti-Rig-I antibody.I SDD-AGE analysis of Mavs aggregates in WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) infected with SeV for 12 h. SDS-PAGE immunoblotting was used as a loading control.J-O qPCR analysis of Ifnβ (J), Ifnα1 (K), Ifnα4 (L), Ifit1 (M), Cxcl10 (N), and Cxcl11 (O) mRNA in WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV (VSV or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without VSV-GFP viruses for 12 h, and viral infectivity was detected by fluorescence microscopy (P)WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without VSV-GFP viruses for 12 h, and viral infectivity was detected by western blot analysis (Q)WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without VSV-GFP viruses for 12 h, and viral infectivity was detected by flow cytometry analysis (n = 3) (R). Data information: All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).