Figure 1B WT and SirT7 - / - mice at 10 days ( top ) and 2 months ( bottom ) of age . C Weight distribution of WT and SirT7 - / - mice ( n = 3 - 10 female mice per time point and genotype ; P value = 0 . 003 by unpaired t - Test ) . Kaplan Meier survival curves ( n = 170 WT and n = 58 SirT7 - / - mice ; Log - rank Test P value < 0 . 0001 ) .

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Figure 1B WT and SirT7-/- mice at 10 days (top) and 2 months (bottom) of age.C Weight distribution of WT and SirT7-/- mice (n=3-10 female mice per time point and genotype; P value = 0.003 by unpaired t-Test).Kaplan Meier survival curves (n=170 WT and n=58 SirT7-/- mice; Log-rank Test P value <0.0001).


Figure 2A Representative 3D reconstructed CT scans showing increased kyphosis in 16 month old SirT7 - / - mice compared with WT . B Representative gonadal fat pads from WT and SirT7 - / - 16 month old mice . C Quantitation of body weight ( left ) and gonadal fat pad mass normalized to total body weight ( right ) ( mean ± SEM ; 4 samples per genotype ) . D IGF - 1 protein levels in serum measured by ELISA ( mean ± SEM ; 3 - 8 mice per genotype and age group ) . E , F Dot plots of Lin - Sca1 + cKit + ( LSK ) cells ( middle and right ) from WT and SirT7 - / - bone marrow cells . Cells were gated for negative staining of lineage markers B220 , CD3 , CD11b , CD19 , Gr - 1 , and Ter - 119 ( Left ) , and analyzed for Sca1 and cKit expression ( middle and right ) . ( F ) Quantitation of ( E ) ( mean ± SEM ; 4 mice per genotype ) . G Bone marrow , thymus , and spleen cell number in young WT and SirT7 - / - mice ( mean ± SEM ; 3 - 5 mice per genotype ) . H mRNA expression of p16 gene normalized to GAPDH measured by RT - PCR from young and old WT and SirT7 - / - fibroblasts ( mean ± SEM ; 3 samples per genotype ) . I , J Senescence - associated β - galactosidasestaining ( I , blue ) in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and grown for 7 days in selection media . ( J ) Quantitation of the number of senescent cells shown in ( I ) ( mean ± SEM ; 3 independent cell lines per genotype ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by ANOVA Single Factor .

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Figure 2A Representative 3D reconstructed CT scans showing increased kyphosis in 16 month old SirT7-/- mice compared with WT.B Representative gonadal fat pads from WT and SirT7-/- 16 month old mice.C Quantitation of body weight (left) and gonadal fat pad mass normalized to total body weight (right) (mean ± SEM; 4 samples per genotype).D IGF-1 protein levels in serum measured by ELISA (mean ± SEM; 3-8 mice per genotype and age group).E, F Dot plots of Lin-Sca1+cKit+ (LSK) cells (middle and right) from WT and SirT7-/- bone marrow cells. Cells were gated for negative staining of lineage markers B220, CD3, CD11b, CD19, Gr-1, and Ter-119 (Left), and analyzed for Sca1 and cKit expression (middle and right). (F) Quantitation of (E) (mean ± SEM; 4 mice per genotype).G Bone marrow, thymus, and spleen cell number in young WT and SirT7-/- mice (mean ± SEM; 3-5 mice per genotype).H mRNA expression of p16 gene normalized to GAPDH measured by RT-PCR from young and old WT and SirT7-/- fibroblasts (mean ± SEM; 3 samples per genotype).I, J Senescence-associated β-galactosidasestaining (I, blue) in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and grown for 7 days in selection media. (J) Quantitation of the number of senescent cells shown in (I) (mean ± SEM; 3 independent cell lines per genotype).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.


Figure 3A , B Dot plots of FACS cell cycle analyses of WT and SirT7 - / - MEFs in passage 3 ( P3 ) and 6 ( P6 ) using EdU incorporation and 7AAD ( A ) . Percentages of cells in Sub G1 ( apoptotic cells , left square ) and cells with DNA content above 4N ( polyploid , right square ) . ( B ) Quantitation of experiment shown in ( A ) ( mean ± SEM ; 3 samples per genotype ) . C Survival curve for WT and SirT7 - / - thymocytes after X - Ray irradiation ( IR ) at the indicated doses . Cell death was quantified by FACS using AnexinV and 7AAD staining 18 hours post insult ( mean ± SEM ; 3 samples per genotype from one of two independent experiments ) . D Clonogenic assays in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and irradiated at the indicated X - Ray doses , then plated at low density . After 9 days , colonies were stained with crystal violet and counted ( mean ± SEM ; 3 independent cell lines per genotype ) . F Quantitation of experiment shown in ( E ) ( mean ± SEM ; 9 WT and 3 SirT7 - / - samples ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by ANOVA Single Factor .

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Figure 3A, B Dot plots of FACS cell cycle analyses of WT and SirT7-/- MEFs in passage 3 (P3) and 6 (P6) using EdU incorporation and 7AAD (A). Percentages of cells in Sub G1 (apoptotic cells, left square) and cells with DNA content above 4N (polyploid, right square). (B) Quantitation of experiment shown in (A) (mean ± SEM; 3 samples per genotype).C Survival curve for WT and SirT7-/- thymocytes after X-Ray irradiation (IR) at the indicated doses. Cell death was quantified by FACS using AnexinV and 7AAD staining 18 hours post insult (mean ± SEM; 3 samples per genotype from one of two independent experiments).D Clonogenic assays in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and irradiated at the indicated X-Ray doses, then plated at low density. After 9 days, colonies were stained with crystal violet and counted (mean ± SEM; 3 independent cell lines per genotype).F Quantitation of experiment shown in (E) (mean ± SEM; 9 WT and 3 SirT7-/- samples).*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.


Figure 4A Quantitation of neutral comet assays , using passage 3 WT and SirT7 - / - primary MEFs , showing ( left ) the amount ( % ) of DNA in the tail and ( right ) the tail moment ( see Fig S3A for representative images ) . B Western blot showing ATM and KAP1 phosphorylation and total protein levels in WT and SirT7 - / - primary fibroblasts after IR ( 8Gy ) . ATM inhibitor ( ATMi ) KU - 55933 was added 30 min prior to irradiation where indicated . One representative blot from 4 independent experiments is shown . C - F IF analysis of WT and SirT7 - / - primary fibroblasts showing γH2AX dynamics after DNA damage induction . Cells were untreated ( NoIR ) or treated with 1Gy of X - rays ( IR ) and fixed at the indicated times post insult . Cells were pulsed with EdU ( green ) 30 min prior to fixation , then stained for γH2AX ( red ) and counterstained with DAPI ( blue ) ( n > 30 cells per group / mice ; 3 mice per genotype ) . ( C ) Representative images of untreated S - phase ( NoIR , EdU positive ) WT and SirT7 - / - nuclei ( scale bar 5µm ) . ( D ) Quantitation of the number of γH2AX foci per nucleus at the indicated time points post IR ( 1Gy ) , and indicated cell cycle phases . ( E ) Quantitation of experiment described in ( C ) showing mean number of γH2AX foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after IR in G2 ( mean ± SEM from 3 independent experiments ) . Total γH2AX foci and γH2AX foci overlapping with or at the periphery of heterochromatic regions were enumerated . Nuclei and pericentric heterochromatin ( chromocenters ) were segmented by DAPI staining . Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number . ( F ) Representative images of WT and SirT7 - / - primary fibroblasts in G2 phase showing γH2AX ( red ) and DAPI ( blue ) at the indicated period of time after IR . ( Right ) 3D rendering of the IF segmentation depicting nuclei ( pale blue ) , chromocenters ( darker blue ) , and γH2AX ( yellow denotes foci associated with pericentric heterochromatin , otherwise foci are red ) . Scale bar 2 µm . G FACS quantitation of WT and SirT7 - / - MEF cells in S - phase after insult with 10 mM hydroxyurea ( HU ) for 24 hours . Cells were fixed and stained with 7AAD and cell cycle was monitored by FACS ( mean ± SEM ; 5 samples per genotype ) . H , I DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7 - / - MEFs . ( H ) Representative images from cells labeled for 20 min with IdU ( green ) followed by 20 min of CldU ( red ) . ( I ) Quantitation of fork velocity ( fiber length / labeling time ; mean ± SEM ; 3 samples per genotype per condition ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by ANOVA Single Factor .

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

Figure 4A Quantitation of neutral comet assays, using passage 3 WT and SirT7-/- primary MEFs, showing (left) the amount (%) of DNA in the tail and (right) the tail moment (see Fig S3A for representative images).B Western blot showing ATM and KAP1 phosphorylation and total protein levels in WT and SirT7-/- primary fibroblasts after IR (8Gy). ATM inhibitor (ATMi) KU-55933 was added 30 min prior to irradiation where indicated. One representative blot from 4 independent experiments is shown.C-F IF analysis of WT and SirT7-/- primary fibroblasts showing γH2AX dynamics after DNA damage induction. Cells were untreated (NoIR) or treated with 1Gy of X-rays (IR) and fixed at the indicated times post insult. Cells were pulsed with EdU (green) 30 min prior to fixation, then stained for γH2AX (red) and counterstained with DAPI (blue) (n>30 cells per group/mice; 3 mice per genotype). (C) Representative images of untreated S-phase (NoIR, EdU positive) WT and SirT7-/- nuclei (scale bar 5µm). (D) Quantitation of the number of γH2AX foci per nucleus at the indicated time points post IR (1Gy), and indicated cell cycle phases. (E) Quantitation of experiment described in (C) showing mean number of γH2AX foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after IR in G2 (mean ± SEM from 3 independent experiments). Total γH2AX foci and γH2AX foci overlapping with or at the periphery of heterochromatic regions were enumerated. Nuclei and pericentric heterochromatin (chromocenters) were segmented by DAPI staining. Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number.(F) Representative images of WT and SirT7-/- primary fibroblasts in G2 phase showing γH2AX (red) and DAPI (blue) at the indicated period of time after IR. (Right) 3D rendering of the IF segmentation depicting nuclei (pale blue), chromocenters (darker blue), and γH2AX (yellow denotes foci associated with pericentric heterochromatin, otherwise foci are red). Scale bar 2 µm.G FACS quantitation of WT and SirT7-/- MEF cells in S-phase after insult with 10 mM hydroxyurea (HU) for 24 hours. Cells were fixed and stained with 7AAD and cell cycle was monitored by FACS (mean ± SEM; 5 samples per genotype).H, I DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7-/- MEFs. (H) Representative images from cells labeled for 20 min with IdU (green) followed by 20 min of CldU (red). (I) Quantitation of fork velocity (fiber length/labeling time; mean ± SEM; 3 samples per genotype per condition).*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.


Figure 5A - E IF analysis of WT and SirT7 - / - primary fibroblasts after IR ( 1Gy ) and 1 hour chase . Cells were pulsed with EdU 30 min prior to fixation , stained with antibodies against γH2AX and 53BP1 and then counterstained with DAPI ( scale bar 5µm ) . ( A ) Representative images from each cell cycle stage showing merge ( top - left ) including EdU ( green ) , 53BP1 ( top - right , magenta ) , γH2AX ( bottom - left , red ) , and 3D rendering of reconstructed Z - stacks with 53BP1 foci modeled as spheres ( bottom - right ; pale - blue : nucleus ; magenta spheres : 53BP1 foci ) . ( B ) Quantitation of the number of 53BP1 foci per nucleus , and ( C ) mean volume of 53BP1 foci ( n > 30 cells per group / mice ; 3 mice per genotype ) . ( D - E ) Same as in ( B - C ) , upon overexpression of SIRT7 WT or catalytically inactive point mutant SIRT7 - H188Y in WT and SirT7 - / - primary fibroblasts . F Detection of endogenous chromatin - bound and nucleoplasmic 53BP1 protein from WT and SirT7 - / - MEFs before and after IR ( 8Gy ) by western blot . Histone H3 was used as loading control , and tubulin as control for fractionation . G Same as ( F ) , using 293T - REX cells treated with doxycycline or vehicle to induce SIRT7 - HA expression . H ChIP - on break assay . ( Top ) Schematic of I - SceI substrate construct introduced into HT1080 cells , which contains a single I - SceI site located within a puromycin resistance cassette . Shown along the top are the coordinates for amplicons probed by Q - PCR . ( Bottom ) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells . Q - PCR measurements were normalized to input DNA and non - I - SceI treated samples ( No Cut ) ( mean ± SEM ; one of two independent experiments shown ) . ( Bottom Right ) western blot demonstrating efficient knockdown of SirT7 , with Histone H3 as a loading control . I , J NHEJ repair assay using GFP expression - based reporter system in SirT7 - depleted HT1080 cells ( SirT7 Knockdown ) versus control ( Scramble Control ) . ( I ) Representative FACS dot plots . ( J ) Quantitation of ( I ) ; mean ± SEM of 3 independent clones per condition . Values are normalized to control ( Scramble Control + I - SceI ) mean . K , L Class - switch recombination in splenic B cells from WT and SirT7 - / - mice stimulated with lipopolysaccharides ( LPS ) and interleukin 4 ( IL4 ) . ( K ) The switching from IgM to IgG1 ( left ) and to IgG3 ( right ) was measured by FACS . ( L ) Quantitation of ( K ) showing mean ± SEM of 3 - 4 samples per genotype . One representative experiment from two is shown . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by ANOVA Single Factor .

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Figure 5A-E IF analysis of WT and SirT7-/- primary fibroblasts after IR (1Gy) and 1 hour chase. Cells were pulsed with EdU 30 min prior to fixation, stained with antibodies against γH2AX and 53BP1 and then counterstained with DAPI (scale bar 5µm). (A) Representative images from each cell cycle stage showing merge (top-left) including EdU (green), 53BP1 (top-right, magenta), γH2AX (bottom-left, red), and 3D rendering of reconstructed Z-stacks with 53BP1 foci modeled as spheres (bottom-right; pale-blue: nucleus; magenta spheres: 53BP1 foci). (B) Quantitation of the number of 53BP1 foci per nucleus, and (C) mean volume of 53BP1 foci (n>30 cells per group/mice; 3 mice per genotype). (D-E) Same as in (B-C), upon overexpression of SIRT7 WT or catalytically inactive point mutant SIRT7-H188Y in WT and SirT7-/- primary fibroblasts.F Detection of endogenous chromatin-bound and nucleoplasmic 53BP1 protein from WT and SirT7-/- MEFs before and after IR (8Gy) by western blot. Histone H3 was used as loading control, and tubulin as control for fractionation.G Same as (F), using 293T-REX cells treated with doxycycline or vehicle to induce SIRT7-HA expression.H ChIP-on break assay. (Top) Schematic of I-SceI substrate construct introduced into HT1080 cells, which contains a single I-SceI site located within a puromycin resistance cassette. Shown along the top are the coordinates for amplicons probed by Q-PCR. (Bottom) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells. Q-PCR measurements were normalized to input DNA and non-I-SceI treated samples (No Cut) (mean ± SEM; one of two independent experiments shown). (Bottom Right) western blot demonstrating efficient knockdown of SirT7, with Histone H3 as a loading control.I, J NHEJ repair assay using GFP expression-based reporter system in SirT7-depleted HT1080 cells (SirT7 Knockdown) versus control (Scramble Control). (I) Representative FACS dot plots. (J) Quantitation of (I); mean ± SEM of 3 independent clones per condition. Values are normalized to control (Scramble Control + I-SceI) mean.K, L Class-switch recombination in splenic B cells from WT and SirT7-/- mice stimulated with lipopolysaccharides (LPS) and interleukin 4 (IL4). (K) The switching from IgM to IgG1 (left) and to IgG3 (right) was measured by FACS. (L) Quantitation of (K) showing mean ± SEM of 3-4 samples per genotype. One representative experiment from two is shown.*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.


Figure 6A , B H3K18Ac IF in WT and SirT7 - / - fibroblasts . Cells were pulsed with EdU ( green ) , stained with antibody against H3K18Ac ( magenta ) and then counterstained with DAPI ( blue ) . ( A ) Representative IF images of late S - phase nuclei ( EdU positive ; scale bar 5µm ) . ( B ) Quantitation of ( A ) throughout the cell cycle . C Same as in ( B ) , in S - phase cells at the indicated times after IR ( 1Gy ) ( mean ± SEM of > 30 cells per cell cycle , time point , and genotype ) . D Laser - induced DNA damage in HT1080 cells expressing HA - tagged SirT7 stained with antibodies against γH2AX ( red ) and HA ( green ) , then counterstained with DAPI ( blue ) 30 min post damage ( scale bar 3 µm ) . E ChIP assays of SIRT7 enrichment at the indicated loci as described in Figure 5H ( mean ± SEM ; one of two independent experiments is shown ) . F Recruitment kinetics of GFP - tagged SirT7 after laser - induced microirradiation . ( Top ) Representative cell at the indicated times after induction of DNA damage ( white circle ; scale bar 2µm ) . Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal . ( Middle ) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU - 55933 ATM inhibitor ( ATMi ) , 10M Olaparib PARP inhibitor ( PARPi ) , or DMSO . KU - 55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage ( mean ± SEM ; sample size : SIRT7 - GFP , n = 34 ; SIRT7 - GFP + ATMi , n = 36 ; SIRT7 - GFP + PARPi = 18 ) . ( Bottom ) Same as ( middle ) except quantitation of SIRT7 - GFP relative intensity within the nucleolus . Data was acquired at five second intervals over a span of five min . G ChIP assays of H3K18Ac enrichment at the indicated loci as described in Figure 5H . H3K18Ac enrichment adjacent to the induced DSB in control and SIRT7 depleted cells ( bottom left ) , or in cells overexpressing SIRT7 in the presence or absence of PARP inhibitors ( PARPi ) ( bottom right ) ( mean ± SEM ; one of two independent experiments is shown ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by ANOVA Single Factor .

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

Figure 6A, B H3K18Ac IF in WT and SirT7-/- fibroblasts. Cells were pulsed with EdU (green), stained with antibody against H3K18Ac (magenta) and then counterstained with DAPI (blue). (A) Representative IF images of late S-phase nuclei (EdU positive; scale bar 5µm). (B) Quantitation of (A) throughout the cell cycle.C Same as in (B), in S-phase cells at the indicated times after IR (1Gy) (mean ± SEM of >30 cells per cell cycle, time point, and genotype).D Laser-induced DNA damage in HT1080 cells expressing HA-tagged SirT7 stained with antibodies against γH2AX (red) and HA (green), then counterstained with DAPI (blue) 30 min post damage (scale bar 3 µm).E ChIP assays of SIRT7 enrichment at the indicated loci as described in Figure 5H (mean ± SEM; one of two independent experiments is shown).F Recruitment kinetics of GFP-tagged SirT7 after laser-induced microirradiation. (Top) Representative cell at the indicated times after induction of DNA damage (white circle; scale bar 2µm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU-55933 ATM inhibitor (ATMi), 10M Olaparib PARP inhibitor (PARPi), or DMSO. KU-55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage (mean ± SEM; sample size: SIRT7-GFP, n=34; SIRT7-GFP + ATMi, n=36; SIRT7-GFP + PARPi=18). (Bottom) Same as (middle) except quantitation of SIRT7-GFP relative intensity within the nucleolus. Data was acquired at five second intervals over a span of five min.G ChIP assays of H3K18Ac enrichment at the indicated loci as described in Figure 5H. H3K18Ac enrichment adjacent to the induced DSB in control and SIRT7 depleted cells (bottom left), or in cells overexpressing SIRT7 in the presence or absence of PARP inhibitors (PARPi) (bottom right) (mean ± SEM; one of two independent experiments is shown).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.


Figure 7A - H NHEJ activity in HT1080 or NIH3T3 cells stably overexpressing H3 - WT , H3 - K18Q or H3 - K18R ( naive , acetylated or deacetylated H3K18 residues , respectively ) . A Random integration assay in HT1080 cells using a linearized pSMCV vector containing a puromycin resistance cassette ( mean number of puromycin resistant colonies normalized by plating efficiency ± SEM ; One representative transfection from 3 is shown ) . B NHEJ repair assay using GFP expression - based reporter system described in Fig 5I ( mean ± SEM of 3 independent experiments . See Fig EV5B for representative FACS dot plots ) . C - D IF staining of NIH3T3 cells after IR ( 1Gy ) and 1 hour chase . Cells were pulsed with EdU 30 min prior to fixation , stained with antibodies against γH2AX and 53BP1 , and counterstained with DAPI . Representative images can be found in Fig EV5C . ( C ) Quantitation of the number of 53BP1 foci per nucleus , and ( D ) mean volume of 53BP1 foci ( mean ± SEM of > 30 cells per condition and cell cycle stage ) . E Western blot analysis of 53BP1 in chromatin and non - chromatin fractions from NIH3T3 cells expressing H3 - WT , H3 - K18Q or H3 - K18R vectors and exposed to IR ( 10Gy ) . One representative blot is shown from 3 independent experiments . Shown is 53BP1 , GAPDH for fractionation control , and H3 for loading control . F - H Same as in ( C - D ) except using antibodies against γH2AX ( red ) and RIF1 ( magenta ) . ( F ) Representative images from cells in S - phase ( EdU positive ; scale bar 5µm ) . ( G ) Quantitation of the number of RIF1 foci per nucleus , and ( H ) mean volume of RIF1 foci ( mean ± SEM of > 30 cells per condition and cell cycle stage ) . I 53BP1 enrichment at the indicated loci as described in Figure 5H in Scramble control or SirT7 knockdown cells expressing the indicated H3 constructs ( mean ± SEM ; one of two independent experiments ) . J Western blot analysis of chromatin fractions from primary WT and SirT7 - / - MEFs expressing the indicated H3 constructs and exposed to IR ( 10Gy ) . Shown are levels of 53BP1 , SIRT7 , H3 - Myc , GAPDH for fractionation control , and H3 for loading control .

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Figure 7A-H NHEJ activity in HT1080 or NIH3T3 cells stably overexpressing H3-WT, H3-K18Q or H3-K18R (naive, acetylated or deacetylated H3K18 residues, respectively).A Random integration assay in HT1080 cells using a linearized pSMCV vector containing a puromycin resistance cassette (mean number of puromycin resistant colonies normalized by plating efficiency ± SEM; One representative transfection from 3 is shown).B NHEJ repair assay using GFP expression-based reporter system described in Fig 5I (mean ± SEM of 3 independent experiments. See Fig EV5B for representative FACS dot plots).C-D IF staining of NIH3T3 cells after IR (1Gy) and 1 hour chase. Cells were pulsed with EdU 30 min prior to fixation, stained with antibodies against γH2AX and 53BP1, and counterstained with DAPI. Representative images can be found in Fig EV5C. (C) Quantitation of the number of 53BP1 foci per nucleus, and (D) mean volume of 53BP1 foci (mean ± SEM of >30 cells per condition and cell cycle stage).E Western blot analysis of 53BP1 in chromatin and non-chromatin fractions from NIH3T3 cells expressing H3-WT, H3-K18Q or H3-K18R vectors and exposed to IR (10Gy). One representative blot is shown from 3 independent experiments. Shown is 53BP1, GAPDH for fractionation control, and H3 for loading control.F-H Same as in (C-D) except using antibodies against γH2AX (red) and RIF1 (magenta). (F) Representative images from cells in S-phase (EdU positive; scale bar 5µm). (G) Quantitation of the number of RIF1 foci per nucleus, and (H) mean volume of RIF1 foci (mean ± SEM of >30 cells per condition and cell cycle stage).I 53BP1 enrichment at the indicated loci as described in Figure 5H in Scramble control or SirT7 knockdown cells expressing the indicated H3 constructs (mean ± SEM; one of two independent experiments).J Western blot analysis of chromatin fractions from primary WT and SirT7-/- MEFs expressing the indicated H3 constructs and exposed to IR (10Gy). Shown are levels of 53BP1, SIRT7, H3-Myc, GAPDH for fractionation control, and H3 for loading control.


Figure 1B : Consequences of ROS on the SUMO proteome vary amongst cell lines . HeLa , MCF7 , U2OS or RPE cells were treated with increasing H2O2 concentration for 15 min and lysed in Laemmli buffer supplemented with 20 mM N - Ethylmaleimide ( NEM ) . Samples were analyzed by immunoblotting with SUMO1 antibodies . C : H2O2 induces loss of TRIM28 SUMOylation in HeLa cells but not in U2OS cells . HeLa or U2OS cells were mock treated or treated with 500 µM H2O2 for 5 or 15 min . Cells were lysed with 1 % SDS in PBS buffer and 20 mM NEM . Upon immunoprecipitation of the SUMO1 proteome , samples were analyzed by immunoblotting with TRIM28 antibodies . D : Induction of the Uba2 ~ Ubc9 disulfide is a conserved mechanism . HeLa , MCF7 , U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min , lysed in non reducing buffer supplemented with 20 mM NEM , and analyzed by immunoblotting with Uba2 antibodies .

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Figure 1B: Consequences of ROS on the SUMO proteome vary amongst cell lines. HeLa, MCF7, U2OS or RPE cells were treated with increasing H2O2 concentration for 15 min and lysed in Laemmli buffer supplemented with 20 mM N-Ethylmaleimide (NEM). Samples were analyzed by immunoblotting with SUMO1 antibodies.C: H2O2 induces loss of TRIM28 SUMOylation in HeLa cells but not in U2OS cells. HeLa or U2OS cells were mock treated or treated with 500 µM H2O2 for 5 or 15 min. Cells were lysed with 1% SDS in PBS buffer and 20 mM NEM. Upon immunoprecipitation of the SUMO1 proteome, samples were analyzed by immunoblotting with TRIM28 antibodies.D: Induction of the Uba2~Ubc9 disulfide is a conserved mechanism. HeLa, MCF7, U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min, lysed in non reducing buffer supplemented with 20 mM NEM, and analyzed by immunoblotting with Uba2 antibodies.


Figure 2A : Upper panel : Strategy for the identification of oxidation resistant Ubc9 variants . Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations . Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2 . For this purpose , we used a FRET - based in vitro SUMOylation assay with recombinant SUMO E1 , YFP - SUMO and CFP - RanGAPtail and ATP . Lower panel : Ubc9 W103R is H2O2 resistant . Bacterial lysates containing Ubc9 wt ( left panel ) or Ubc9 W103R ( right panel ) were tested as described . B : Ubc9 W103 mutants are H2O2 resistant but not fully active . Left panel : Recombinant Ubc9 W103R , W103A and W103F were purified ( embedded panel ) . Resistance against oxidation was tested under conditions of limiting E1 enzyme : 21 nM Aos1 / Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP - SUMO1 and CFP - RanGAPtail . Right panel : To compare specific activities of wt and mutants in the absence of H2O2 , sumoylation assays were carried out using limiting Ubc9 concentration . Reactions contained 35 nM Aos1 / Uba2 , 11 nM Ubc9 , 85 nM each of YFP - SUMO1 and CFP - RanGAPtail and 1 mM ATP .

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Figure 2A: Upper panel: Strategy for the identification of oxidation resistant Ubc9 variants. Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations. Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2. For this purpose, we used a FRET-based in vitro SUMOylation assay with recombinant SUMO E1, YFP-SUMO and CFP-RanGAPtail and ATP. Lower panel: Ubc9 W103R is H2O2 resistant. Bacterial lysates containing Ubc9 wt (left panel) or Ubc9 W103R (right panel) were tested as described.B: Ubc9 W103 mutants are H2O2 resistant but not fully active. Left panel: Recombinant Ubc9 W103R, W103A and W103F were purified (embedded panel). Resistance against oxidation was tested under conditions of limiting E1 enzyme: 21 nM Aos1/Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP-SUMO1 and CFP-RanGAPtail. Right panel: To compare specific activities of wt and mutants in the absence of H2O2, sumoylation assays were carried out using limiting Ubc9 concentration. Reactions contained 35 nM Aos1/Uba2, 11 nM Ubc9, 85 nM each of YFP-SUMO1 and CFP-RanGAPtail and 1 mM ATP.


Figure 3B : In FRET - based assay , Ubc9 D100A is active upon H2O2 treatment . Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig . 2B . C : Ubc9 wt and D100A are equally active in CFP - RanGAPtail SUMOylation . Assays were with 35 nM Aos1 / Uba2 and 15 nM Ubc9 . D : Ubc9 D100A is fully competent to form a thioester with SUMO . Single turnover reactions were performed using 630 nM E1 , 3 , 3 µM SUMO1 and 600 nM Ubc9 . Reactions were stopped at different time points by addition of non - reducing buffer . . E : Ubc9 wt and D100A are equally active in PIAS2b - dependent SUMOylation . Assays were with 300 nM GST - p53 , 170 nM E1 , 120 nM Ubc9 , 5 µM SUMO2 and 49 nM GST - Pias2bF : Ubc9 wt or D100A are equally active in RanBP2 - dependent SUMOylation . Assays were with 650 nM YFP - SP100 , 110 nM of E1 , 25 Ubc9 , 40 nM RanBP2ΔFG and 3 , 5 µM SUMO1 ( Left panel ) or SUMO2 ( Right panel ) .

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Figure 3B: In FRET-based assay, Ubc9 D100A is active upon H2O2 treatment. Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig. 2B.C: Ubc9 wt and D100A are equally active in CFP-RanGAPtail SUMOylation. Assays were with 35 nM Aos1/Uba2 and 15 nM Ubc9.D: Ubc9 D100A is fully competent to form a thioester with SUMO. Single turnover reactions were performed using 630 nM E1, 3,3 µM SUMO1 and 600 nM Ubc9. Reactions were stopped at different time points by addition of non-reducing buffer..E: Ubc9 wt and D100A are equally active in PIAS2b - dependent SUMOylation. Assays were with 300 nM GST-p53, 170 nM E1, 120 nM Ubc9, 5 µM SUMO2 and 49 nM GST-Pias2bF: Ubc9 wt or D100A are equally active in RanBP2 - dependent SUMOylation. Assays were with 650 nM YFP-SP100, 110 nM of E1, 25 Ubc9, 40 nM RanBP2ΔFG and 3,5 µM SUMO1 (Left panel) or SUMO2 (Right panel).


Figure 4A : Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT . 21 nM Aos1 / Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP - SUMO1 and CFP - RanGAPtail diluted in DTT - free buffer . The final DTT concentration in the reaction was 6 µM . Prior to addition of ATP , extra amount of DTT was added at indicated final concentration . B : Both Uba2 ~ Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A . 100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2 . One hour after H2O2 addition , 500 µM reduced glutathione was added . Samples were taken at indicated time points and the Uba2 ~ Ubc9 disulfide was monitored by immunoblotting against Uba2 . Lower panel : Quantification of the ratio between crosslinked Uba2 vs total

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

Figure 4A: Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT. 21 nM Aos1/Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP-SUMO1 and CFP-RanGAPtail diluted in DTT-free buffer. The final DTT concentration in the reaction was 6 µM. Prior to addition of ATP, extra amount of DTT was added at indicated final concentration.B: Both Uba2~Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A.100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2. One hour after H2O2 addition, 500 µM reduced glutathione was added. Samples were taken at indicated time points and the Uba2~Ubc9 disulfide was monitored by immunoblotting against Uba2. Lower panel: Quantification of the ratio between crosslinked Uba2 vs total Uba2


Figure 5A : Generation of polyclonal populations of stable cells . Top panel : Replacement strategy : U2OS cells transfected with pIRES - hrGFP II - based vectors were selected via addition of G418 . After 4 weeks , low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks . Bottom panel : Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9 : U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected . Cells were lysed in Laemmli buffer and Ubc9 and a - tubulin as loading control were detected by immunoblotting . B : Ubc9 D100A is severely impaired for disulfide bond formation with Uba2 . Stable U2OS cells were transfected with siRNA against Ubc9 for 72h , treated with 250 µM H2O2 for different time points , from 2 min to 2 hours , lysed in non - reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide ( upper panel ) or free Uba2 as loading control ( see also Appendix Fig . S5 ) . Lower panel : relative intensities of Ubc9 bands were measured using imageJ . Highest intensities in both cell population were set to 1 . C : Ubc9 D100A impairs cell survival upon H2O2 treatment . Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h , transferred into 6 well plates ( 20 000 cells / well ) and treated with increasing H2O2 concentration for 1h . Ten days later , cells were washed with PBS , fixed with 4 % formaldehyde and stained with crystal violet . The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin . An arbitrary value of 1 was given to the area occupied by non - treated cells . The graph is showing the mean of 4 independent experiments . Error bars are S . E . M . * : p < 0 . 05D : Constitutive expression of Ubc9 D100A affects cellgrowth . 103 cells were plated per 10 cm plate and cultivated for 10 days in 20 % or 5 % O2 incubators . Subsequently , cells were colored using crystal violet . The area of each clone was measured using ImageJ . Representative plates are shown in the upper panel . Lower left panel : Distribution of colony sizes , shown for one of the three biological replicates . Lower right panel : Comparison of the colonies size between " Ubc9 wt " and " Ubc9 DA " cells . The average size of " Ubc9 wt " was set to 1 . Shown is the mean of 3 independent experiments , each of which was performed in technical triplicates . Error bars are S . E . M .

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Figure 5A: Generation of polyclonal populations of stable cells. Top panel: Replacement strategy: U2OS cells transfected with pIRES-hrGFP II-based vectors were selected via addition of G418. After 4 weeks, low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks. Bottom panel: Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9: U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected. Cells were lysed in Laemmli buffer and Ubc9 and a-tubulin as loading control were detected by immunoblotting.B: Ubc9 D100A is severely impaired for disulfide bond formation with Uba2. Stable U2OS cells were transfected with siRNA against Ubc9 for 72h, treated with 250 µM H2O2 for different time points, from 2 min to 2 hours, lysed in non-reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide (upper panel) or free Uba2 as loading control (see also Appendix Fig. S5). Lower panel: relative intensities of Ubc9 bands were measured using imageJ. Highest intensities in both cell population were set to 1.C: Ubc9 D100A impairs cell survival upon H2O2 treatment. Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h, transferred into 6 well plates (20 000 cells / well) and treated with increasing H2O2 concentration for 1h. Ten days later, cells were washed with PBS, fixed with 4% formaldehyde and stained with crystal violet. The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin. An arbitrary value of 1 was given to the area occupied by non-treated cells. The graph is showing the mean of 4 independent experiments. Error bars are S.E.M. *: p < 0.05D: Constitutive expression of Ubc9 D100A affects cellgrowth. 103 cells were plated per 10 cm plate and cultivated for 10 days in 20% or 5% O2 incubators. Subsequently, cells were colored using crystal violet. The area of each clone was measured using ImageJ. Representative plates are shown in the upper panel. Lower left panel: Distribution of colony sizes, shown for one of the three biological replicates. Lower right panel: Comparison of the colonies size between "Ubc9 wt"and "Ubc9 DA" cells. The average size of "Ubc9 wt" was set to 1. Shown is the mean of 3 independent experiments, each of which was performed in technical triplicates. Error bars are S.E.M.


Figure 6A : Phosphorylation of ATM is impaired in Ubc9 D100A cells . Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies . B : Phosphorylation of gamma - H2AX is impaired in Ubc9 D100A . Experiment was performed as in A . Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies . C : Phosphorylation of Chk2 is impaired in Ubc9 D100A . Experiment was performed as described in A . Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies . D : Ubc9 D100A cells are fully competent for phosphorylation of ATM upon hydroxyurea exposure . Stable U2OS cell populations were exposed to 2 . 5 mM hydroxyurea or 250 µM H2O2 . Cells were lysed at indicated time points and analysed by immunoblotting with the indicated antibodies . E : Phosphorylation of Chk1 is not impaired in Ubc9 D100A cells . Stable U2OS cells were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2 . 5 mM hydroxyurea . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies .

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Figure 6A: Phosphorylation of ATM is impaired in Ubc9 D100A cells. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.B: Phosphorylation of gamma-H2AX is impaired in Ubc9 D100A. Experiment was performed as in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.C: Phosphorylation of Chk2 is impaired in Ubc9 D100A. Experiment was performed as described in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.D: Ubc9 D100A cells are fully competent for phosphorylation of ATM upon hydroxyurea exposure. Stable U2OS cell populations were exposed to 2.5 mM hydroxyurea or 250 µM H2O2. Cells were lysed at indicated time points and analysed by immunoblotting with the indicated antibodies.E: Phosphorylation of Chk1 is not impaired in Ubc9 D100A cells. Stable U2OS cells were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2.5 mM hydroxyurea. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


Figure 7A : Impaired y - H2AX and 53BP1 accumulation in Ubc9 D100A cells . Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h . Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed . Left panel : representative immunofluorescence images . Right panel : quantification of cells with γ - H2AX and 53BP1 foci . Error bars represent S . E . M , n = 3 independent experiments * p < 0 . 05 . B : H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells . Cells were treated as described in A . Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h . Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed . An alkaline comet assay was performed to evaluate the DNA damage . Representative images are shown . Error intervals represent S . E . M , n = 4 independent experiments .

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Figure 7A: Impaired y-H2AX and 53BP1 accumulation in Ubc9 D100A cells. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. Left panel: representative immunofluorescence images. Right panel: quantification of cells with γ-H2AX and 53BP1 foci. Error bars represent S.E.M, n=3 independent experiments * p < 0.05.B: H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells. Cells were treated as described in A. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. An alkaline comet assay was performed to evaluate the DNA damage. Representative images are shown. Error intervals represent S.E.M, n=4 independent experiments.


Figure 8A : Diamide is inducing the SUMO E1 ~ E2 disulfide but not ATM phosphorylation . U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2 . Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer . The lysates were resolved by SDS - PAGE . B : Left panel : The PP2A inhibitor okadaic acid rescues the y - H2AX defect of the " Ubc9 DA " cells . " Ubc9 wt " or " Ubc9 DA " cells were treated with 0 . 25 µM okadaic acid for 30 min . After addition of 250 µM H2O2 in fresh medium for 3 hours , immunofluorescence microscopy was performed as described in Fig . 7A . Error bars represent S . E . M , n = 3 independent experiments . Right panel : Representative immunofluorescence images . C : Left panel : Ubc9 D100A enhances sensitivity to Ara - C or etoposide . Cell survival assays were performed as described in Fig . 5C . Cells were exposed to Ara - C or etoposide ( VP16 ) for 1 h and evaluated 10 days later . Right panel : N - acetylcysteine treatment rescues the higher toxicity of Ara - C or etoposide on " Ubc9 DA " cells . Cells were treated with 0 . 5 mM NAC 16 h prior to addition of etoposide or Ara - C . Graphs represent means of three to four independent experiments , error bars represent S . E . M . * p < 0 . 05 .

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Figure 8A: Diamide is inducing the SUMO E1~E2 disulfide but not ATM phosphorylation. U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2. Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer. The lysates were resolved by SDS-PAGE.B: Left panel: The PP2A inhibitor okadaic acid rescues the y-H2AX defect of the "Ubc9 DA" cells. "Ubc9 wt" or "Ubc9 DA" cells were treated with 0.25 µM okadaic acid for 30 min. After addition of 250 µM H2O2 in fresh medium for 3 hours, immunofluorescence microscopy was performed as described in Fig. 7A. Error bars represent S.E.M, n=3 independent experiments. Right panel: Representative immunofluorescence images.C: Left panel: Ubc9 D100A enhances sensitivity to Ara-C or etoposide. Cell survival assays were performed as described in Fig. 5C. Cells were exposed to Ara-C or etoposide (VP16) for 1 h and evaluated 10 days later. Right panel: N-acetylcysteine treatment rescues the higher toxicity of Ara-C or etoposide on "Ubc9 DA" cells. Cells were treated with 0.5 mM NAC 16 h prior to addition of etoposide or Ara-C. Graphs represent means of three to four independent experiments, error bars represent S.E.M. * p < 0.05.


figf1 ( b ) IRGMC mRNA was significantly enriched in miR - 196B complexes . Extracts of cells expressing FLAG - tagged - AGO1 and transfected with biotinylated miR - 196B or control miR - 20 as indicated and then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification ( immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads ) . IRGM mRNA variants were quantified using quantitative RT - PCR ; results are presented as the ratio between miR - 196B and miR - 20 ( non - relevant miRNA ) pull - downs and the mean of three independent experiments ± standard deviation ( s . d . ) . IP strep , immunoprecipitation straptavidine . ( c ) HEK293 cells ( IRGMC / C ) were transfected with either FLAG - tagged IRGMC or FLAG - tagged IRGMT plasmids and co - transfected with miR - 196B . Immunoblotting with an IRGM antibody revealed the specificity of the downregulation effect mediated by the miRNA IRGM mRNA interaction . Quantification of the immunoblot signals are presented as IRGM expression relative to actin ( mean of at least three independent experiments ± s . d . ) .

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figf1(b) IRGMC mRNA was significantly enriched in miR-196B complexes. Extracts of cells expressing FLAG-tagged-AGO1 and transfected with biotinylated miR-196B or control miR-20 as indicated and then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification (immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads). IRGM mRNA variants were quantified using quantitative RT-PCR; results are presented as the ratio between miR-196B and miR-20 (non-relevant miRNA) pull-downs and the mean of three independent experiments ± standard deviation (s.d.). IP strep, immunoprecipitation straptavidine.(c) HEK293 cells (IRGMC/C) were transfected with either FLAG-tagged IRGMC or FLAG-tagged IRGMT plasmids and co-transfected with miR-196B. Immunoblotting with an IRGM antibody revealed the specificity of the downregulation effect mediated by the miRNA IRGM mRNA interaction. Quantification of the immunoblot signals are presented as IRGM expression relative to actin (mean of at least three independent experiments ± s.d.).


figf2 ( a ) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls ( n = 40 ) or individuals with Crohn ' s disease ( CD ) ( n = 67 ) without or with active inflammation and labeled for miR - 196A . ( L , lumen ; LP , lamina propria ) . Scale bars in the upper panel , 25 μm ; scale bars in the lower panel , 10 μm . ( b ) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls ( n = 8 ) or individuals with Crohn ' s disease with no inflammation ( n = 16 ) , quiescent ( defined as low - grade inflammation ) ( n = 8 ) or acute inflammation ( n = 8 ) using laser capture microdissection . After RNA extraction , miR - 196A ( black bars ) and miR - 196B ( white bars ) relative expression was analyzed using RNU19 , 44 and U6 . To overcome possible inter - individual bias , the lamina propria fraction value was used for relative quantification . Due to high differences in expression between healthy and inflamed tissues , the results are presented as a log2 - fold ratio . Error bars indicate the s . d . of the ΔΔCt values . ( c ) Representative in situ staining for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn ' s disease with a defined genotype in a healthy non - inflamed or an acute inflamed phase . Scale bars , 32 μm . ( d ) Mean ( black line ) , s . e . m . ( white box ) and 95 % CI of the mean of the IRGM expression level for 40 healthy subjects ( 32 individuals with C / C and 8 individuals with C / T ) and 67 individuals with Crohn ' s disease ( 45 with C / C and 22 with C / T ) with quiescent or inflamed colon mucosa . We performed statistical analysis using ANOVA ( P = 0 . 0015 ) and an unpaired Student ' s t - test ( the one tail P value is indicated on the figure ) .

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figf2(a) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls (n = 40) or individuals with Crohn's disease (CD) (n = 67) without or with active inflammation and labeled for miR-196A. (L, lumen; LP, lamina propria). Scale bars in the upper panel, 25 μm; scale bars in the lower panel, 10 μm.  (b) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls (n = 8) or individuals with Crohn's disease with no inflammation (n = 16), quiescent (defined as low-grade inflammation) (n = 8) or acute inflammation (n = 8) using laser capture microdissection. After RNA extraction, miR-196A (black bars) and miR-196B (white bars) relative expression was analyzed using RNU19, 44 and U6. To overcome possible inter-individual bias, the lamina propria fraction value was used for relative quantification. Due to high differences in expression between healthy and inflamed tissues, the results are presented as a log2-fold ratio. Error bars indicate the s.d. of the ΔΔCt values.(c) Representative in situ staining for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn's disease with a defined genotype in a healthy non-inflamed or an acute inflamed phase. Scale bars, 32 μm. (d) Mean (black line), s.e.m. (white box) and 95% CI of the mean of the IRGM expression level for 40 healthy subjects (32 individuals with C/C and 8 individuals with C/T) and 67 individuals with Crohn's disease (45 with C/C and 22 with C/T) with quiescent or inflamed colon mucosa. We performed statistical analysis using ANOVA (P = 0.0015) and an unpaired Student's t-test (the one tail P value is indicated on the figure).


figf3 ( a ) The basal flux of autophagy is affected by IRGM expression level . HEK293 cells transfected with an IRGM - expressing plasmid , miR - 196 or siIRGM were treated with bafilomycin A1 for 2 h and processed for immunoblotting with anti - LC3B . ( b ) Quantification of LC3 - II relative to actin ( mean of three independent experiments ± s . d . ) . ( c ) Downregulation of IRGM expression by miR - 196 abrogates AIEC - mediated autophagy in cells treated with autophagic inhibitors ( Inh ) or transfected with miR - 196B and infected for 4 h with AIEC LF82 ( mean of three independent experiments ± s . d . ) . ( d ) Confocal microscopic examination of LC3 revealed a significant decrease in the percentage of LC3 - associated ( red ) LF82 bacteria ( green ) in miR - 196 transfected cells compared to control cells ( mean ± s . d . ) . ( e ) miR - 196 transfection leads to increased intracellular LF82 replication . Results are expressed as a fold increase ± s . e . m . of intracellular bacteria . ( f ) IRGM overexpression did not inhibit autophagic flux and it increased LC3 - II accumulation slightly in response to AIEC infection . HEK293 cells were transfected with IRGM - expressing plasmid , treated with autophagic inhibitors and infected with AIEC bacteria for 4 h ( mean of three independent experiments ± s . d . ) . ( g ) Confocal microscopic examination showed an increased percentage of LC3 - associated AIEC bacteria in IRGM cells compared to control cells ( mean ± s . d . ) . ( h ) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria . ( i ) Most of the bacteria reside in non - acidic vacuoles , as shown with lysotracker at 8 h post infection ( means of three independent experiments ± s . d . ) .

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figf3(a) The basal flux of autophagy is affected by IRGM expression level. HEK293 cells transfected with an IRGM-expressing plasmid, miR-196 or siIRGM were treated with bafilomycin A1 for 2 h and processed for immunoblotting with anti-LC3B. (b) Quantification of LC3-II relative to actin (mean of three independent experiments ± s.d.).(c) Downregulation of IRGM expression by miR-196 abrogates AIEC-mediated autophagy in cells treated with autophagic inhibitors (Inh) or transfected with miR-196B and infected for 4 h with AIEC LF82 (mean of three independent experiments ± s.d.).(d) Confocal microscopic examination of LC3 revealed a significant decrease in the percentage of LC3-associated (red) LF82 bacteria (green) in miR-196 transfected cells compared to control cells (mean ± s.d.).(e) miR-196 transfection leads to increased intracellular LF82 replication. Results are expressed as a fold increase ± s.e.m. of intracellular bacteria.(f) IRGM overexpression did not inhibit autophagic flux and it increased LC3-II accumulation slightly in response to AIEC infection. HEK293 cells were transfected with IRGM-expressing plasmid, treated with autophagic inhibitors and infected with AIEC bacteria for 4 h (mean of three independent experiments ± s.d.).(g) Confocal microscopic examination showed an increased percentage of LC3-associated AIEC bacteria in IRGM cells compared to control cells (mean ± s.d.). (h) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria. (i) Most of the bacteria reside in non-acidic vacuoles, as shown with lysotracker at 8 h post infection (means of three independent experiments ± s.d.).


figf1 ( a ) Kinetics of the appearance of P14 cells in spleens of C57BL / 6 mice given adoptive transfer of 1 × 104 P14 cells , monitored after infection of recipients with LCMV Armstrong strain . ( b ) Flow cytometry of cell size at various stages of P14 differentiation , assessed by forward scatter ( FSC ) . ( c ) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation . Data are representative of two independent experiments with samples pooled from 5 - 20 mice for each time point .

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figf1(a) Kinetics of the appearance of P14 cells in spleens of C57BL/6 mice given adoptive transfer of 1 × 104 P14 cells, monitored after infection of recipients with LCMV Armstrong strain.(b) Flow cytometry of cell size at various stages of P14 differentiation, assessed by forward scatter (FSC).(c) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation. Data are representative of two independent experiments with samples pooled from 5-20 mice for each time point.


figf2 ( b ) Flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a , assessed on days 5 , 8 and 30 after infection of recipients with LCMV . Numbers in blue outlined areas indicate percent GFP − cells among transduced ( Thy - 1 . 1 + ) P14 cells . ( c ) Summary of results in b , showing autophagy activity as percent GFP − cells . * P ≤ 0 . 0005 ( unpaired t - test ) . ( d ) Multispectral - imaging flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a , assessed on day 8 after infection , showing GFP signals from GFP + transduced ( CD45 . 1 + Thy - 1 . 1 + ) P14 cells . Original magnification , × 60 . ( e ) Summary of results in d , presented as the frequency of cells with more than one GFP + punctum among vector transduced Thy - 1 . 1 + P14 cells . Each symbol represents an individual mouse ; small horizontal lines indicate the mean ( ± s . e . m . ) . Data are representative of three independent experiments ( b , d ) or three independent experiments with three or more mice per group ( e ; error bars , s . e . m . ) or six mice per group ( c ; error bars , s . e . m . ) .

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figf2(b) Flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5, 8 and 30 after infection of recipients with LCMV. Numbers in blue outlined areas indicate percent GFP− cells among transduced (Thy-1.1+) P14 cells. (c) Summary of results in b, showing autophagy activity as percent GFP− cells. *P ≤ 0.0005 (unpaired t-test).(d) Multispectral-imaging flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on day 8 after infection, showing GFP signals from GFP+ transduced (CD45.1+ Thy-1.1+) P14 cells. Original magnification, ×60. (e) Summary of results in d, presented as the frequency of cells with more than one GFP+ punctum among vector transduced Thy-1.1+ P14 cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). Data are representative of three independent experiments (b,d) or three independent experiments with three or more mice per group (e; error bars, s.e.m.) or six mice per group (c; error bars, s.e.m.).


figf3 ( b ) Flow cytometry of splenocytes from recipient mice ( n = 6 ) given adoptive transfer of P14 cells transduced with retrovirus as in a , assessed on days 5 and 8 after infection of recipients with LCMV ( left ) , and quantification of P14 cells per spleen ( right ) . Numbers adjacent to outlined areas ( left ) indicate percent P14 ( CD45 . 1 + ) cells . * P ≤ 0 . 0005 ( unpaired t - test ) . ( c ) Flow cytometry of the size of P14 cells transduced with wild - type mCherry - GFP - LC3b retrovirus ( assessed as in Fig . 1b ) in mice treated as in b , assessed at days 5 and 8 after infection . ( d ) Efficiency of retroviral transduction ( left ) and expression of GFP and mCherry ( right ) in retrovirus - transduced P14 cells , assessed by flow cytometry gated on CD45 . 1 + P14 cells ( left ) or CD45 . 1 + Thy - 1 . 1 + P14 cells ( right ) . Numbers in quadrants indicate percent CD45 . 1 + Thy - 1 . 1 − ( untransduced ) P14 cells ( top left ) or CD45 . 1 + Thy - 1 . 1 + ( transduced ) P14 cells ( top right ; magenta ) ( left ) or percent cells in each ( right ) . ( e ) Ratio of the geometric mean fluorescence intensity ( gMFI ) of GFP to that of mCherry ( GFP / mCherry ) for the cells in d ; each symbol represents an individual mouse ; numbers above lines connecting groups indicate difference in ratio . Data are representative of two independent experiments with three or more mice per group ( error bars ( b ) , s . e . m . ) .

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

figf3(b) Flow cytometry of splenocytes from recipient mice (n = 6) given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5 and 8 after infection of recipients with LCMV (left), and quantification of P14 cells per spleen (right). Numbers adjacent to outlined areas (left) indicate percent P14 (CD45.1+) cells. *P ≤ 0.0005 (unpaired t-test).(c) Flow cytometry of the size of P14 cells transduced with wild-type mCherry-GFP-LC3b retrovirus (assessed as in Fig. 1b) in mice treated as in b, assessed at days 5 and 8 after infection.(d) Efficiency of retroviral transduction (left) and expression of GFP and mCherry (right) in retrovirus-transduced P14 cells, assessed by flow cytometry gated on CD45.1+ P14 cells (left) or CD45.1+Thy-1.1+ P14 cells (right). Numbers in quadrants indicate percent CD45.1+Thy-1.1− (untransduced) P14 cells (top left) or CD45.1+Thy-1.1+ (transduced) P14 cells (top right; magenta) (left) or percent cells in each (right). (e) Ratio of the geometric mean fluorescence intensity (gMFI) of GFP to that of mCherry (GFP/mCherry) for the cells in d; each symbol represents an individual mouse; numbers above lines connecting groups indicate difference in ratio. Data are representative of two independent experiments with three or more mice per group (error bars (b), s.e.m.).


figf4 ( a ) Genomic deletion of Atg7 in H - 2Db - gp33 + T cells and in total activated ( CD44hiCD62Llo ) CD8 + T cells from Atg7fl / fl mice ( Cre − ) and Atg7fl / flGzmb - Cremice ( Cre + ) at day 8 after infection with LCMV Armstrong strain ; results are presented relative to those of Atg7fl / fl mice , set as 100 % . Each symbol represents an individual mouse ; small horizontal lines indicate the mean ( ± s . e . m . ) . ( b ) Immunoblot analysis of Atg7 and the products of its enzymatic activity ( left margin ) in CD44hiCD62Llo CD8 + T cells isolated at day 8 after infection as in a ; β - actin serves as a loading control . ( c ) Flow cytometry of T cells in the peripheral blood at day 8 after infection as in a , gated on CD8 + T cells ( left ) , and of the H - 2Db - gp33 + T cells identified at left , identifying terminal effector ( KLRG1hiCD127lo ) and memory precursor ( KLRG1loCD127hi ) populations ( right ) . Numbers adjacent to outlined areas ( left ) indicate percent H - 2Db - gp33 + T cells ; numbers in quadrants ( right ) indicate percent cells in each . ( d ) Total antigen - specific CD8 + T cells in the spleen , liver and lungs at day 8 after infection as in a . ( e ) Production of IFN - γ and TNF in cells obtained 8 d after infection and stimulated ex vivo with various peptides ( above plots ) , gated on total CD8 + T cells . Numbers in quadrants indicate percent cells in each . ( f ) Flow cytometry of CD8 + T cells in the peripheral blood of mice at day 15 after infection as in a ( numbers in plots as in c , left ) . ( g ) Quantification of tetramer - positive cells in spleens at day 15 after infection as in a . ( h ) Kinetics of the appearance H - 2Db - gp33 + CD8 + T cells among peripheral blood mononuclear cells ( PBMCs ) after infection as in a ( left ) ; each line represents one mouse . Right , frequency of H - 2Db - gp33 + CD8 + T cells remaining on day 15 relative to that on day 8 , set as 100 % . ( i ) Quantification of tetramer - positive cells at day 30 after infection as in a . Horizontal dashed lines ( g , h ) indicate the limit of detection . * P ≤ 0 . 05 , * * P ≤ 0 . 005 and * * * P ≤ 0 . 0005 ( unpaired t - test ) . Data were pooled from two independent experiments ( a ) or at least two independent experiments ( c - i ) with three to seven mice per group ( error bars , s . e . m . ) or are from one experiment with samples obtained from two independent experiments ( b ) .

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

figf4(a) Genomic deletion of Atg7 in H-2Db-gp33+ T cells and in total activated (CD44hiCD62Llo) CD8+ T cells from Atg7fl/fl mice (Cre −) and Atg7fl/flGzmb-Cremice (Cre +) at day 8 after infection with LCMV Armstrong strain; results are presented relative to those of Atg7fl/fl mice, set as 100%. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.).(b) Immunoblot analysis of Atg7 and the products of its enzymatic activity (left margin) in CD44hiCD62Llo CD8+ T cells isolated at day 8 after infection as in a; β-actin serves as a loading control.(c) Flow cytometry of T cells in the peripheral blood at day 8 after infection as in a, gated on CD8+ T cells (left), and of the H-2Db-gp33+ T cells identified at left, identifying terminal effector (KLRG1hiCD127lo) and memory precursor (KLRG1loCD127hi) populations (right). Numbers adjacent to outlined areas (left) indicate percent H-2Db-gp33+ T cells; numbers in quadrants (right) indicate percent cells in each.(d) Total antigen-specific CD8+ T cells in the spleen, liver and lungs at day 8 after infection as in a.(e) Production of IFN-γ and TNF in cells obtained 8 d after infection and stimulated ex vivo with various peptides (above plots), gated on total CD8+ T cells. Numbers in quadrants indicate percent cells in each.(f) Flow cytometry of CD8+ T cells in the peripheral blood of mice at day 15 after infection as in a (numbers in plots as in c, left).(g) Quantification of tetramer-positive cells in spleens at day 15 after infection as in a.(h) Kinetics of the appearance H-2Db-gp33+ CD8+ T cells among peripheral blood mononuclear cells (PBMCs) after infection as in a (left); each line represents one mouse. Right, frequency of H-2Db-gp33+ CD8+ T cells remaining on day 15 relative to that on day 8, set as 100%.(i) Quantification of tetramer-positive cells at day 30 after infection as in a. Horizontal dashed lines (g,h) indicate the limit of detection. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data were pooled from two independent experiments (a) or at least two independent experiments (c-i) with three to seven mice per group (error bars, s.e.m.) or are from one experiment with samples obtained from two independent experiments (b).


figf5 ( a ) Flow cytometry of circulating LCMV - specific CD8 + T cells among peripheral blood mononuclear cells from Atg5fl / fl and Atg5fl / flGzmb - Cremice on days 8 , 15 and 50 after infection with LCMV Armstrong strain . Numbers adjacent to outlined areas indicate percent H - 2Db - gp33 + T cells , gated on CD8 + T cells . ( b ) Longitudinal kinetics of the appearance of H - 2Db - gp33 + T cells in the peripheral blood of mice as in a ( left ) , and percent H - 2Db - gp33 + CD8 + T cells remaining on day 15 ( right ) ( all presented as in Fig . 4h ) . ( c ) Flow cytometry of T cells in tissues on day 279 after infection as in a ( numbers in plots as in a , among total gated CD8 + T cells ) . ( d ) Total T cells specific for H - 2Db - gp33 ( left ) or H - 2Db - NP396 ( right ) in the spleen , liver and lungs of mice as in a . * P ≤ 0 . 05 , * * P ≤ 0 . 005 and * * * P ≤ 0 . 0005 ( unpaired t - test ) . Data are representative of two independent experiments with three to seven mice per group ( error bars ( d ) , s . e . m . ) .

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

figf5(a) Flow cytometry of circulating LCMV-specific CD8+ T cells among peripheral blood mononuclear cells from Atg5fl/fl and Atg5fl/flGzmb-Cremice on days 8, 15 and 50 after infection with LCMV Armstrong strain. Numbers adjacent to outlined areas indicate percent H-2Db-gp33+ T cells, gated on CD8+ T cells. (b) Longitudinal kinetics of the appearance of H-2Db-gp33+ T cells in the peripheral blood of mice as in a (left), and percent H-2Db-gp33+ CD8+ T cells remaining on day 15 (right) (all presented as in Fig. 4h).(c) Flow cytometry of T cells in tissues on day 279 after infection as in a (numbers in plots as in a, among total gated CD8+ T cells). (d) Total T cells specific for H-2Db-gp33 (left) or H-2Db-NP396 (right) in the spleen, liver and lungs of mice as in a. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to seven mice per group (error bars (d), s.e.m.).


figf6 ( a , b ) Flow cytometry of CD8 + T cell obtained from chimeras generated ( as in Supplementary Fig . 5a ) by reconstitution of wild - type mice with a mixture of bone marrow cells from Atg7fl / fl C57BL / 6 and wild - type C57BL / 6C57BL / 6 ( Atg7fl / fl vs C57BL / 6 ) or from Atg7fl / flGzmb - Cremice and wild - type C57BL / 6mice and ( Atg7fl / flGzmb - Cre vs C57BL / 6 ) , gated ( far left ) as populations specific for H - 2Db - gp33 ( a ) or H - 2Db - NP396 ( b ) and then assessed , on days 8 , 15 and 30 after infection of recipients with LCMV Armstrong infection , as CD45 . 2 + ( Atg7fl / fl or Atg7fl / flGzmb - Cre ) donor cells or CD45 . 1 + ( C57BL / 6 ) donor cells ( middle ) . Far right , appearance of tetramer - positive T cells ( key , donor source ) in the peripheral blood of chimeras from day 8 to day 30 after infection , presented relative to that at day 8 after infection , set as 100 % . ( c ) Frequency of CD45 . 2 + antigen - specific CD8 + T cells in the spleen , liver and lungs on day 30 after infectionwith LCMV Armstrongas in a , b . Data are representative of two independent experiments with three to four mice per group ( error bars , s . e . m . ) .

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

figf6(a,b) Flow cytometry of CD8+ T cell obtained from chimeras generated (as in Supplementary Fig. 5a) by reconstitution of wild-type mice with a mixture of bone marrow cells from Atg7fl/fl C57BL/6 and wild-type C57BL/6C57BL/6 (Atg7fl/fl vs C57BL/6) or from Atg7fl/flGzmb-Cremice and wild-type C57BL/6mice and (Atg7fl/flGzmb-Cre vs C57BL/6), gated (far left) as populations specific for H-2Db-gp33 (a) or H-2Db-NP396 (b) and then assessed, on days 8, 15 and 30 after infection of recipients with LCMV Armstrong infection, as CD45.2+ (Atg7fl/fl or Atg7fl/flGzmb-Cre) donor cells or CD45.1+ (C57BL/6) donor cells (middle). Far right, appearance of tetramer-positive T cells (key, donor source) in the peripheral blood of chimeras from day 8 to day 30 after infection, presented relative to that at day 8 after infection, set as 100%.(c) Frequency of CD45.2+ antigen-specific CD8+ T cells in the spleen, liver and lungs on day 30 after infectionwith LCMV Armstrongas in a,b. Data are representative of two independent experiments with three to four mice per group (error bars, s.e.m.).


figf8 ( a ) Kinetics of the appearance of H - 2Db - gp276 + T cells in the peripheral blood of Atg7fl / fl and Atg7fl / flGzmb - Cre mice at various times ( horizontal axis ) after infection with LCMV clone 13 . ( b ) Total IFN - γ - secreting cells in spleens obtained from mice at day 8 or day 15 after infection as in a , assessed after 5 h of stimulation ex vivo with various peptides ( horizontal axes ) . ( c ) Viral titers in serum of mice at various times ( horizontal axis ) after infection with LCMV clone 13 as in a . Each symbol represents an individual mouse ; small horizontal lines indicate the mean ( ± s . e . m . ) . * P ≤ 0 . 05 , * * P ≤ 0 . 005 and * * * P ≤ 0 . 0005 ( unpaired t - test ) . Data are representative of two independent experiments with three to five mice per group ( error bars , s . e . m . ) .

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

figf8(a) Kinetics of the appearance of H-2Db-gp276+ T cells in the peripheral blood of Atg7fl/fl and Atg7fl/flGzmb-Cre mice at various times (horizontal axis) after infection with LCMV clone 13.(b) Total IFN-γ-secreting cells in spleens obtained from mice at day 8 or day 15 after infection as in a, assessed after 5 h of stimulation ex vivo with various peptides (horizontal axes).(c) Viral titers in serum of mice at various times (horizontal axis) after infection with LCMV clone 13 as in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to five mice per group (error bars, s.e.m.).


Figure 1B : Single - cell growth of drug - treated cells as in A as a function of time since MreB - motion stop according to panel A . Left and middle : Relative increase ( left ) and single - cell rates ( middle ) of volume , mass , surface , and length . Gray asterisks indicate single - cell mass at lysis . Right : Relative changes of dry - mass density , surface - to - mass ratio , surface - to - volume ratio , and width . D : Complex nutrient shift of non - dividing cells ( S382 ) growing on agarose pad ( MM + 0 . 02 % glucose ) in the presence of vancomycin ( 100 μg / mL ) . Same quantities as in B , as a function of time since MreB - motion stop . Cells were transiently starved for glucose ( through addition of 0 . 5 % ( w / v ) αMG and 0 . 25 % ( w / v ) 2DG ) , followed by addition of excess glucose ( 1 % ) . Different from A - C , all compounds were added to the agarose pad as droplets and reached cells by diffusion .

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

Figure 1B: Single-cell growth of drug-treated cells as in A as a function of time since MreB-motion stop according to panel A. Left and middle: Relative increase (left) and single-cell rates (middle) of volume, mass, surface, and length. Gray asterisks indicate single-cell mass at lysis. Right: Relative changes of dry-mass density, surface-to-mass ratio, surface-to-volume ratio, and width.D: Complex nutrient shift of non-dividing cells (S382) growing on agarose pad (MM+0.02% glucose) in the presence of vancomycin (100 μg/mL). Same quantities as in B, as a function of time since MreB-motion stop. Cells were transiently starved for glucose (through addition of 0.5% (w/v) αMG and 0.25% (w/v) 2DG), followed by addition of excess glucose (1%). Different from A-C, all compounds were added to the agarose pad as droplets and reached cells by diffusion.


Figure 2A : Single - cell growth of non - dividing b42 cells ( ΔmepS / pBAD30 - mepS ) grown on LB agarose pads as a function of time after addition of arabinose ( 0 . 2 % ) in form of a droplet . Left : Relative increase of volume , mass , surface , and length . Middle : Elongation rate and surface - growth rate . Right : Relative changes of surface - to - mass ratio and width .

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

Figure 2A: Single-cell growth of non-dividing b42 cells (ΔmepS/pBAD30-mepS) grown on LB agarose pads as a function of time after addition of arabinose (0.2%) in form of a droplet. Left: Relative increase of volume, mass, surface, and length. Middle: Elongation rate and surface-growth rate. Right: Relative changes of surface-to-mass ratio and width.


Figure 3B - C : Cell bending : B : Time - lapses of bending filamentous cells ( S290 ) in donut microchambers ( 3 % agarose ; RDM + glu ) and grown without ( top ) or with ( bottom ) D - cycloserine ( 1 mM ) as a function of time since D - cycloserine treatment , starting > 10 min after D - cycloserine treatment ( the time of MreB - motion stop D - cycloserine - treated cells were osmotically shocked ( NaCl ) after 33 min of treatment , which releases a fraction of cells C : Change of cell centerline curvature as a function of cell length between 10 min after start of D - cycloserine treatment and release of cells through osmotic shockD - E : Cell straightening . D : Time - lapses of straightening filamentous cells ( S290 ) previously bent in donuts : control ( top ) and D - cycloserine treated cells ( bottom ) as a function of time since simultaneous release from donuts and beginning of drug treatment . E : Change of centerline curvature as a function of relative length from 10 min after D - cycloserine treatment ( time of MreB - motion stop ) .

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Figure 3B-C: Cell bending: B: Time-lapses of bending filamentous cells (S290) in donut microchambers (3% agarose; RDM+glu) and grown without (top) or with (bottom) D-cycloserine (1 mM) as a function of time since D-cycloserine treatment, starting >10 min after D-cycloserine treatment (the time of MreB-motion stop D-cycloserine-treated cells were osmotically shocked (NaCl) after 33 min of treatment, which releases a fraction of cells C: Change of cell centerline curvature as a function of cell length between 10 min after start of D-cycloserine treatment and release of cells through osmotic shockD-E: Cell straightening. D: Time-lapses of straightening filamentous cells (S290) previously bent in donuts: control (top) and D-cycloserine treated cells (bottom) as a function of time since simultaneous release from donuts and beginning of drug treatment. E: Change of centerline curvature as a function of relative length from 10 min after D-cycloserine treatment (time of MreB-motion stop).


Figure 4C : Relative change of cell width of non - dividing cells ( S257 ) grown in LB during combined D - cycloserine treatment ( 5 mM ) and a hypo - osmotic ramp as in A ( DCS + Hypo ramp ) , as compared to cells exposed to the hypo - osmotic ramp only ( Hypo ramp ) or treated with D - cycloserine only ( DCS ) . D - cycloserine was contained in both agarose pads , and treatment started 15 min prior to the shock .

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Figure 4C: Relative change of cell width of non-dividing cells (S257) grown in LB during combined D-cycloserine treatment (5 mM) and a hypo-osmotic ramp as in A (DCS + Hypo ramp), as compared to cells exposed to the hypo-osmotic ramp only (Hypo ramp) or treated with D-cycloserine only (DCS). D-cycloserine was contained in both agarose pads, and treatment started 15 min prior to the shock.


Figure 1Comparable mean SV number per ribbon in wild - type at the distal ( Ds . , H ) and the proximal ( Pr . , I ) part of the ribbon . More SVs are observed at inhibitory OtofPga / Pga ribbons compared to inhibitory wild - type synapses at both ribbon parts , and further at OtofPga / Pga synapses under rest in the Pr . part . Data are presented as mean ± SEM ; the red dotted line highlights the wild - type mean SV number per ribbon under rest as a reference point ; Representative virtual sections of wild - type ( J ) and OtofPga / Pga ( K ) ribbon synapses under inhibition . Counted RA - SVs are marked with white asterisks . Magnification , 12 , 000x ; scale bar , 100 nm . Exemplary virtual sections of wild - type and OtofPga / Pga under inhib ( L , M ) , rest ( L ' , M ' ) and stim ( L ' ' , M ' ' ) . At OtofPga / Pga SVs are densely packed . Blue arrowheads indicate proteinaceous filaments between wild - type SVs . Magnification 12 , 000x ; scale bar , 100 nm .

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

Figure 1Comparable mean SV number per ribbon in wild-type at the distal (Ds., H) and the proximal (Pr., I) part of the ribbon. More SVs are observed at inhibitory OtofPga/Pga ribbons compared to inhibitory wild-type synapses at both ribbon parts, and further at OtofPga/Pga synapses under rest in the Pr. part. Data are presented as mean ± SEM; the red dotted line highlights the wild-type mean SV number per ribbon under rest as a reference point;Representative virtual sections of wild-type (J) and OtofPga/Pga (K) ribbon synapses under inhibition. Counted RA-SVs are marked with white asterisks. Magnification, 12,000x; scale bar, 100 nm.Exemplary virtual sections of wild-type and OtofPga/Pga under inhib (L,M), rest (L',M') and stim (L'',M''). At OtofPga/Pga SVs are densely packed. Blue arrowheads indicate proteinaceous filaments between wild-type SVs. Magnification 12,000x; scale bar, 100 nm.


Figure 2Illustrations show the two major SVs associated with filament / s in the wild - type RA - SV pool at rest ; synaptic ribbon ( R , red ) , SVs ( dark green ) with filament / s ( blue ) . Not drawn to scale . ( A , B ) Ri - attached SVs ( Ri . ) and ( C , D ) interconnected SVs ( In . ) harbor single ( A , C ) or multiple ( B , D ) filaments . Example virtual section from resting wild - type ; single Ri . ( A ' , A ' ' ) and In . ( C ' , C ' ' ) , multiple Ri . ( B ' , B ' ' ) and In . ( D ' , D ' ' ) . Magnification 12 , 000x ; scale bar , 40 nm . Structures are color - coded similarly to the illustrations , the number of ribbon - attached filaments per SV are indicated with red arrowhead / s and interconnectors per SV with purple arrowhead / s in A ' ' , B ' ' , C ' ' and D ' ' . Magnification 12 , 000x ; scale bar , 40 nm . The graph is representing the percentage of RA - SVs around the whole ribbon at wild - type under inhib , rest and stim . Upon stimulation , the percentage of SVs without filaments and ribbon - attached SVs are significantly reduced , while interconnected SVs increased significantly .

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

Figure 2Illustrations show the two major SVs associated with filament/s in the wild-type RA-SV pool at rest; synaptic ribbon (R, red), SVs (dark green) with filament/s (blue). Not drawn to scale. (A,B) Ri-attached SVs (Ri.) and (C,D) interconnected SVs (In.) harbor single (A,C) or multiple (B,D) filaments. Example virtual section from resting wild-type; single Ri. (A',A'') and In. (C',C''), multiple Ri. (B',B'') and In. (D',D''). Magnification 12,000x; scale bar, 40 nm. Structures are color-coded similarly to the illustrations, the number of ribbon-attached filaments per SV are indicated with red arrowhead/s and interconnectors per SV with purple arrowhead/s in A'', B'', C'' and D''. Magnification 12,000x; scale bar, 40 nm.The graph is representing the percentage of RA-SVs around the whole ribbon at wild-type under inhib, rest and stim. Upon stimulation, the percentage of SVs without filaments and ribbon-attached SVs are significantly reduced, while interconnected SVs increased significantly.