D 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 ) .

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

D 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 .

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

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

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).


A - 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 .

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

A - 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 ) .

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

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 .

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

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 .

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

E - 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 .

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

F - 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 O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A - 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 .

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

A - 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 .

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

O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM I-ORGANISM O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C - 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 .

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

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 .

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

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 O B-ORGANISM O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-ORGANISM O O O O B-TISSUE I-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O

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.


A - 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 .

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

A - 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 .

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

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

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

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

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 .

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

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

F - 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.


A - 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 .

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

A - 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 .

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

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 .

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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 O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E - 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).


B - 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 .

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

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

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.


A - 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 .

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

A - 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 ) .

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

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 .

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

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 .

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

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

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.


IFN - 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 ) .

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

IFN-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 ) .

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

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 ) .

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 ) .

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

I 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 ) .

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

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 ) .

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

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 ) .

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 ) .

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 ) .

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 ) .

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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).


A 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 .

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A 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 .

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

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 .

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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 .

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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 .

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

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.


A - 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 .

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

A-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 .

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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 .

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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 .

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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 .

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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 .

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

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 .

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

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

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.


A - 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 ) .

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

A-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 ) .

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

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 ) .

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

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 ) .

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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 ) .

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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 .

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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 ) .

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

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 )

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

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

B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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 was detected by flow cytometry analysis (n = 3) (Q).


A - 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 ) .

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

A-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 ) .

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

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 .

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

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 .

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

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 ) .

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

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 )

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

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 )

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

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

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).


qPCR analysis of Ifnβ ( A ) , Ifnα1 ( B ) , Ifnα4 ( C ) , Ifit1 ( D ) , Rig - I ( E ) , Cxcl10 ( F ) mRNA in WT or Sirt5 - deficient BMDC 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 ) .

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

qPCR analysis of Ifnβ (A), Ifnα1 (B), Ifnα4 (C), Ifit1 (D), Rig-I (E), Cxcl10 (F) mRNA in WT or Sirt5-deficient BMDC 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).


qPCR analysis of Cxcl11 ( G ) mRNA in WT or Sirt5 - deficient BMDC 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 ) .

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

qPCR analysis of Cxcl11 (G) mRNA in WT or Sirt5-deficient BMDC 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).


qPCR analysis of Ifnβ ( H ) , Ifnα1 ( I ) mRNA in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without VSV viruses ( 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 ) .

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

qPCR analysis of Ifnβ (H), Ifnα1 (I) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV viruses (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).


qPCR analysis of Ifnα4 ( J ) , Ifit1 ( K ) , Rig - I ( L ) mRNA in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without VSV viruses ( 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 ) .

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

qPCR analysis of Ifnα4 (J), Ifit1 (K), Rig-I (L) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV viruses (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).


qPCR analysis of Cxcl10 ( M ) and Cxcl11 ( N ) mRNA in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without VSV viruses ( 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 ) .

qPCR analysis of Cxcl10 (M) and Cxcl11 (N) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV viruses (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).


O Confocal fluorescence microscopy image of replication in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) after infected with VSV - GFP viruses for 12 h ( MOI = 1 ) ( fluorescence , upper ; bright - field , bottom ) . Scale bar = 75 µm .

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

O Confocal fluorescence microscopy image of replication in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) after infected with VSV-GFP viruses for 12 h (MOI = 1) (fluorescence, upper; bright-field, bottom). Scale bar = 75 µm.


A - B VSV infection increased Mavs succinylation in mouse livers ( A ) and lungs ( B ) . Proteins in the livers ( A ) and lungs ( B ) of WT littermates injected intraperitoneally with ( + ) VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) or PBS control ( - ) ( n = 3 per group ) were detected with the indicated antibodies . Mavs succinylation was determined by anti - succ - K7 - MAVS antibody . Relative succinylation level was quantified ( right panel ) .

O O O B-ORGANISM B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O B-TISSUE O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O

A-B VSV infection increased Mavs succinylation in mouse livers (A) and lungs (B). Proteins in the livers (A) and lungs (B) of WT littermates injected intraperitoneally with (+) VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control (-) (n = 3 per group) were detected with the indicated antibodies. Mavs succinylation was determined by anti-succ-K7-MAVS antibody. Relative succinylation level was quantified (right panel).


C - D Knockout of Sirt5 increased Mavs succinylation in mouse livers ( C ) and lungs ( D ) upon VSV infection . Proteins in the livers ( C ) and lungs ( D ) of Sirt5 - deficient or WT littermates ( Sirt5 - / - or Sirt5 + / + ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) ) ( n = 3 per group ) for 24 h were detected with the indicated antibodies . Mavs succinylation was determined by anti - succ - K7 - MAVS antibody . Relative succinylation level was quantified ( right panel ) .

O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O B-TISSUE O O O O B-ORGANISM B-EXP_ASSAY O O O O B-TISSUE O O O O B-TISSUE O O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O

C-D Knockout of Sirt5 increased Mavs succinylation in mouse livers (C) and lungs (D) upon VSV infection. Proteins in the livers (C) and lungs (D) of Sirt5-deficient or WT littermates (Sirt5 -/- or Sirt5 +/+) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse)) (n = 3 per group) for 24 h were detected with the indicated antibodies. Mavs succinylation was determined by anti-succ-K7-MAVS antibody. Relative succinylation level was quantified (right panel).


E Survival ( Kaplan - Meier curve ) of WT mice ( n = 7 ) and Sirt5 - deficient mice ( n = 7 ) injected intraperitoneally with a high dose of VSV ( 5 × 107 PFU per mouse ) and monitored for 10 d .

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

E Survival (Kaplan-Meier curve) of WT mice (n = 7) and Sirt5-deficient mice (n = 7) injected intraperitoneally with a high dose of VSV (5 × 107 PFU per mouse) and monitored for 10 d.


F ELISA assay of Ifnβ in serum from the WT mice ( n = 4 ) and Sirt5 - deficient mice ( n = 4 ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) ) or PBS control for 24 h . Data information : The graphs represent fold - induction relative to the untreated WT mice . PBS , phosphate buffer saline . 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 or two - way ANOVA analysis ) .

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

F ELISA assay of Ifnβ in serum from the WT mice (n = 4) and Sirt5-deficient mice (n = 4) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse)) or PBS control for 24 h. Data information: The graphs represent fold-induction relative to the untreated WT mice. PBS, phosphate buffer saline. 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 or two-way ANOVA analysis).


G Hematoxylin - and - eosin - stained images of lung sections from mice in F . Scale bar = 50 µm .

O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-TISSUE O O B-ORGANISM O O O O O O O O O

G Hematoxylin-and-eosin-stained images of lung sections from mice in F. Scale bar = 50 µm.


qPCR analysis of Ifnβ ( H ) , Ifnα1 ( I ) , Ifit1 ( J ) , Isg15 ( K ) , Cxcl10 ( L ) , Cxcl11 ( M ) mRNA in the lungs of WT or Sirt5 - deficient mice ( Sirt5 + / + or Sirt5 - / - ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) or PBS control for 24 h . Data information : The graphs represent fold - induction relative to the untreated WT mice . PBS , phosphate buffer saline . 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 or two - way ANOVA analysis ) .

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

qPCR analysis of Ifnβ (H), Ifnα1 (I), Ifit1 (J), Isg15 (K), Cxcl10 (L), Cxcl11 (M) mRNA in the lungs of WT or Sirt5-deficient mice (Sirt5 +/+ or Sirt5 -/-) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control for 24 h. Data information: The graphs represent fold-induction relative to the untreated WT mice. PBS, phosphate buffer saline. 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 or two-way ANOVA analysis).


qPCR analysis of Rig - I ( N ) , and Irf7 ( O ) mRNA in the lungs of WT or Sirt5 - deficient mice ( Sirt5 + / + or Sirt5 - / - ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) or PBS control for 24 h . Data information : The graphs represent fold - induction relative to the untreated WT mice . PBS , phosphate buffer saline . 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 or two - way ANOVA analysis ) .

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

qPCR analysis of Rig-I (N), and Irf7 (O) mRNA in the lungs of WT or Sirt5-deficient mice (Sirt5 +/+ or Sirt5 -/-) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control for 24 h. Data information: The graphs represent fold-induction relative to the untreated WT mice. PBS, phosphate buffer saline. 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 or two-way ANOVA analysis).


P qPCR analysis of VSV mRNA in the lungs of WT or Sirt5 - deficient mice ( Sirt5 + / + or Sirt5 - / - ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) or PBS control for 24 h . Data information : The graphs represent fold - induction relative to the untreated WT mice . PBS , phosphate buffer saline . 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 or two - way ANOVA analysis ) .

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

P qPCR analysis of VSV mRNA in the lungs of WT or Sirt5-deficient mice (Sirt5 +/+ or Sirt5 -/-) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control for 24 h. Data information: The graphs represent fold-induction relative to the untreated WT mice. PBS, phosphate buffer saline. 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 or two-way ANOVA analysis).