Figure 6Immortalized mouse fibroblasts were transduced with a lentiviral vector expressing different mDia2 - targeting shRNAs ( shmDia2 # 1 - 3 ) or empty vector ( sh - EV ) . The knock - down was confirmed by qRT - PCR ( N = 3 ) , Western blot and immunofluorescence . Ki67 staining of mDia2 knock - down and control mouse fibroblasts . N = 3 . migration in scratch assays of mDia2 knock - down and control mouse fibroblasts . N = 3 . qRT - PCR for mDia1 , mDia3 , Fmn1 and Fmn2 and several CAF genes relative to Rps29 using RNA from control ( sh - EV ) and mDia2 knock - down ( sh - mDia2 ) fibroblasts . N = 3 . Representative images of mDia2 knockdown and control mouse fibroblasts stained for p53 ( left ) , and quantification of cells showing accumulation of p53 in the nucleus ( right ) . N = 3 . Representative Western blots for p53 , vinculin and histone H1 using nuclear and cytoplasmic fractions and total lysate of mDia2 knock - down ( F ) or activin A - treated fibroblasts ( 6h , 20 ng / ml ) ( G ) and control mouse fibroblasts and quantification of the nuclear p53 / histone H1 ratio . Vinculin and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions , respectively . N = 6 . Representative Western blots for p53 , mDia2 and GAPDH using total lysates of mDia2 knock - down and control mouse fibroblasts treated for 24 h with pifithrin - α ( PFT - α ) ( 15 μM ) . Representative p53 immunofluorescence images of mDia2 knock - down mouse fibroblasts treated with PFT - α or vehicle ( DMSO ) ( top ) and quantification of cells showing accumulation of p53 in the nucleus ( bottom ) . N = 3 . qRT - PCR for mDia2 , Postn , Mmp13 , Fn1 , Spp1 and Acta2 relative to Rps29 using RNA from mDia2 knock - down and control fibroblasts treated with PFT - α or vehicle . N = 3 . Colony size of SCC13 Act cells ( clone 2 ) after co - culture with either immortalized mDia2 knockdown ( sh - mDia2 ) or control ( sh - EV ) mouse fibroblasts . N = 4 . Representative pictures of 5 - week - old tumors formed upon intradermal co - injection of SCC13 Act cells ( clone 2 ) with sh - EV or sh - mDia2 knock - down immortalized mouse fibroblasts . Graph shows tumor volume at different timepoints of tumor development . N = 3 - 5 tumors per group . Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 Act ( clone 2 ) co - injected with sh - EV or sh - mDia2 fibroblasts , stained for E - cadherin ( green ) and periostin or p53 ( red ) , counterstained with Hoechst ( blue ) .

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Figure 6Immortalized mouse fibroblasts were transduced with a lentiviral vector expressing different mDia2-targeting shRNAs (shmDia2 #1-3) or empty vector (sh-EV). The knock-down was confirmed by qRT-PCR (N=3), Western blot and immunofluorescence.Ki67 staining of mDia2 knock-down and control mouse fibroblasts. N=3.migration in scratch assays of mDia2 knock-down and control mouse fibroblasts. N=3.qRT-PCR for mDia1, mDia3, Fmn1 and Fmn2 and several CAF genes relative to Rps29 using RNA from control (sh-EV) and mDia2 knock-down (sh-mDia2) fibroblasts. N=3.Representative images of mDia2 knockdown and control mouse fibroblasts stained for p53 (left), and quantification of cells showing accumulation of p53 in the nucleus (right). N=3.Representative Western blots for p53, vinculin and histone H1 using nuclear and cytoplasmic fractions and total lysate of mDia2 knock-down (F) or activin A-treated fibroblasts (6h, 20 ng/ml) (G) and control mouse fibroblasts and quantification of the nuclear p53/histone H1 ratio. Vinculin and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. N=6.Representative Western blots for p53, mDia2 and GAPDH using total lysates of mDia2 knock-down and control mouse fibroblasts treated for 24 h with pifithrin-α (PFT-α) (15 μM).Representative p53 immunofluorescence images of mDia2 knock-down mouse fibroblasts treated with PFT-α or vehicle (DMSO) (top) and quantification of cells showing accumulation of p53 in the nucleus (bottom). N=3.qRT-PCR for mDia2, Postn, Mmp13, Fn1, Spp1 and Acta2 relative to Rps29 using RNA from mDia2 knock-down and control fibroblasts treated with PFT-α or vehicle. N=3.Colony size of SCC13 Act cells (clone 2) after co-culture with either immortalized mDia2 knockdown (sh-mDia2) or control (sh-EV) mouse fibroblasts. N=4.Representative pictures of 5-week-old tumors formed upon intradermal co-injection of SCC13 Act cells (clone 2) with sh-EV or sh-mDia2 knock-down immortalized mouse fibroblasts. Graph shows tumor volume at different timepoints of tumor development. N=3-5 tumors per group.Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 Act (clone 2) co-injected with sh-EV or sh-mDia2 fibroblasts, stained for E-cadherin (green) and periostin or p53 (red), counterstained with Hoechst (blue).


Figure 7Tumor volume at different time points after co - injection of SCC13 cells with EV or dnActRIB fibroblasts . N = 5 tumors . Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 cells co - injected with EV or dnActRIB fibroblasts stained for K14 ( left ) or PDGFRα ( right ) ( green ) and mDia2 ( red ) , counterstained with Hoechst ( blue ) . Bar graph shows the percentage of mDia2 - positive area . N = 3 . SCC13 EV or SCC13 Fst cells were injected into the ear skin of NOD / SCID mice and tumor weight was determined 12 weeks after injection . N = 9 - 10 tumors . Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 EV or SSC13 Fst cells stained for K14 or mDia2 ( red ) , counterstained with Hoechst ( blue ) . Bar graph shows the percentage of mDia2 - positive area . N = 3 . Tumors formed by SCC13 Act cells were treated ex vivo for 6h with follistatin ( FST ) ( 50 ng / ml ) and / or SMIFH2 ( 10 μM ) , or with activin A ( 20 ng / ml ) and stained for Ki67 . N = 3 tumor explants , n = 4 histological sections . Representative images of spheroids formed by SCC13 Act cells labeled with PKH67 ( green ) with primary human fibroblasts embedded in collagen gels and graph showing increase in the spheroid area between day 0 and day 3 . Spheroids were either treated with FST ( 50 ng / ml ) or SMIFH2 ( 10 μM ) or left untreated . N = 6 - 8 tumor spheroids . Increase in A431 tumor volume ( mm3 ) compared to day 0 ( time when the tumors were first injected with FST , approximately 3 - 4 weeks after injection of the cells ) upon treatment of the tumors with FST ( 100 ng per injection ) or vehicle ( PBS ) . N = 3 - 5 tumors per group . Representative immunofluorescence images the FST or vehicle - treated tumors , stained for mDia2 ( green ) and K14 ( red ) , counterstained with Hoechst ( blue ) .

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Figure 7Tumor volume at different time points after co-injection of SCC13 cells with EV or dnActRIB fibroblasts. N=5 tumors.Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 cells co-injected with EV or dnActRIB fibroblasts stained for K14 (left) or PDGFRα (right) (green) and mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.SCC13 EV or SCC13 Fst cells were injected into the ear skin of NOD/SCID mice and tumor weight was determined 12 weeks after injection. N=9-10 tumors.Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 EV or SSC13 Fst cells stained for K14 or mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.Tumors formed by SCC13 Act cells were treated ex vivo for 6h with follistatin (FST) (50 ng/ml) and/or SMIFH2 (10 μM), or with activin A (20 ng/ml) and stained for Ki67. N=3 tumor explants, n=4 histological sections.Representative images of spheroids formed by SCC13 Act cells labeled with PKH67 (green) with primary human fibroblasts embedded in collagen gels and graph showing increase in the spheroid area between day 0 and day 3. Spheroids were either treated with FST (50 ng/ml) or SMIFH2 (10 μM) or left untreated. N=6-8 tumor spheroids.Increase in A431 tumor volume (mm3) compared to day 0 (time when the tumors were first injected with FST, approximately 3-4 weeks after injection of the cells) upon treatment of the tumors with FST (100 ng per injection) or vehicle (PBS). N=3-5 tumors per group. Representative immunofluorescence images the FST or vehicle-treated tumors, stained for mDia2 (green) and K14 (red), counterstained with Hoechst (blue).


Figure 1 ( A ) C57BL / 6 mice were inoculated with 6x105 B16Bl6 parental or dnTNF - R1 cells on day 0 and treated daily with 7µg mTNF p . l . from day 10 . Tumor growth is shown as mean tumor size index ( TSI ) + SEM ( n = 6 ) . The line under the graph represents the treatment period . ( B ) B16Bl6 tumor growth after daily p . l . treatment with 7µg mTNF in C57BL / 6J wild - type ( WT ) , TNF - R1 - / - , conditional TNF - R1 reactivation knockout ( p55cneo / cneo ) , or p55cneo / cneo mice with Cre expression in endothelium ( Flk1Cre ) or all cells ( DelCre ) . Tumor growth is shown as mean TSI + SEM ( n = 7 for WT and Flk1Cre , 8 for DelCre , 9 for TNF - R1 - / - and 10 for p55cneo / cneo ) . The line under the graph represents the treatment period . ( C ) PCR analysis for the detection of wild - type , conditional knockout ( cneo ) or reactivated ( lox ) TNF - R1 allele in whole tissues , lung endothelial cells ( LECs ) and lung single - cell suspension depleted of CD31 + cells . ( D ) Toxicity of a single i . v . bolus injection of the indicated dose of mTNF . Mean rectal body temperature + SEM and cumulative survival rates are shown ( n = 4 for Flk1Cre 10µg ; 6 for DelCre 10µg , Flk1Cre 10µg and p55cneo / cneo 15µg ; 7 for WT 10µg ; 9 for WT 8µg , 10 for DelCre 20µg ; and 5 for all other groups ) . For continuity of the temperature graphs , dead mice were included with a temperature of 20 ° C .

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Figure 1(A) C57BL/6 mice were inoculated with 6x105 B16Bl6 parental or dnTNF-R1 cells on day 0 and treated daily with 7µg mTNF p.l. from day 10. Tumor growth is shown as mean tumor size index (TSI) + SEM (n = 6). The line under the graph represents the treatment period.(B) B16Bl6 tumor growth after daily p.l. treatment with 7µg mTNF in C57BL/6J wild-type (WT), TNF-R1-/-, conditional TNF-R1 reactivation knockout (p55cneo/cneo), or p55cneo/cneo mice with Cre expression in endothelium (Flk1Cre) or all cells (DelCre). Tumor growth is shown as mean TSI + SEM (n = 7 for WT and Flk1Cre, 8 for DelCre, 9 for TNF-R1-/- and 10 for p55cneo/cneo). The line under the graph represents the treatment period.(C) PCR analysis for the detection of wild-type, conditional knockout (cneo) or reactivated (lox) TNF-R1 allele in whole tissues, lung endothelial cells (LECs) and lung single-cell suspension depleted of CD31+ cells.(D) Toxicity of a single i.v. bolus injection of the indicated dose of mTNF. Mean rectal body temperature + SEM and cumulative survival rates are shown (n = 4 for Flk1Cre 10µg; 6 for DelCre 10µg, Flk1Cre 10µg and p55cneo/cneo 15µg; 7 for WT 10µg; 9 for WT 8µg, 10 for DelCre 20µg; and 5 for all other groups). For continuity of the temperature graphs, dead mice were included with a temperature of 20°C.


Figure 2 ( B , C ) Viability of L929 parental or mCD20 - expressing cells after 72h stimulation with mTNF or sc mTNF mutant Y86F fused to a BcII10 or mCD20 VHH ( B ) and viability of MCF7 parental or hCD20 - expressing cells after 72h stimulation with hTNF or sc hTNF mutant Y87F fused to a BcII10 or hCD20 VHH ( C ) . Cell viability was measured via an ATP luminescence assay . Each point is the mean of three replicates and error bars are SEM . ( D ) IL - 8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of stimulus , measured via ELISA .

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Figure 2(B,C) Viability of L929 parental or mCD20-expressing cells after 72h stimulation with mTNF or sc mTNF mutant Y86F fused to a BcII10 or mCD20 VHH (B) and viability of MCF7 parental or hCD20-expressing cells after 72h stimulation with hTNF or sc hTNF mutant Y87F fused to a BcII10 or hCD20 VHH (C). Cell viability was measured via an ATP luminescence assay. Each point is the mean of three replicates and error bars are SEM.(D) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of stimulus, measured via ELISA.


Figure 3 ( A ) Toxicity of wt sc mTNF and mutant Y86F after injection ( ↑ ) in naïve C56BL / 6 mice . First injection was i . v . , other injections were i . p . Mean rectal body temperature ± SEM and cumulative survival rates are shown ( n = 4 ) . For continuity of the temperature graphs , dead mice were included with a temperature of 20 ° C . ( B ) Plasma IL - 6 concentrations measured via ELISA . Blood samples were taken 6 hours after i . v . injection of wt sc mTNF or mutant Y86F . ( C ) Immunohistochemical staining for PECAM - 1 ( red ) , ICAM - 1 ( green ) and DNA ( blue ) of B16Bl6 tumors after p . l . treatment with 7µg wt mTNF or 50µg of mCD13 - AFR . Scale bar is 50µm . ( D ) Expression of ICAM - 1 , E - selectin and VEGF - R2 in B16Bl6 tumor 6h after treatment , by qPCR analysis of whole tumor RNA . Error bars are SEM . ns , non - significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 by one - way ANOVA with Bonferroni ' s multiple comparison test . ( E ) Tumor growth and body weight change after daily p . l . injection of indicated ( equimolar ) doses of mCD13 - AFR , non - targeted AFR ( BcII10 - AFR ) or mCD13 VHH control ( CD13 - hIFNα2 ) in the B16Bl6 melanoma model . The line under the graph represents the treatment period . Tumor growth is shown as mean TSI , error bars are SEM ( n = 5 ) . * * * , p < 0 . 001 by two - way ANOVA with Bonferroni ' s multiple comparison test . ( F ) B16Bl6 tumor growth after daily p . l . treatment with indicated doses of mCD13 - AFR , Wortmannin ( Wm ) , Birinapant ( Bir ) or combinations thereof . Wortmannin and Birinapant were given 1 hour before CD13 - AFR . Tumor growth is shown as mean TSI + SEM ( n = 4 for Wm and Bir , 5 for other groups ) . The line under the graph represents the treatment period . TSI of individual tumors of the indicated treatment groups at day 17 after tumor inoculation .

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Figure 3(A) Toxicity of wt sc mTNF and mutant Y86F after injection (↑) in naïve C56BL/6 mice. First injection was i.v., other injections were i.p. Mean rectal body temperature ± SEM and cumulative survival rates are shown (n = 4). For continuity of the temperature graphs, dead mice were included with a temperature of 20°C.(B) Plasma IL-6 concentrations measured via ELISA. Blood samples were taken 6 hours after i.v. injection of wt sc mTNF or mutant Y86F.(C) Immunohistochemical staining for PECAM-1 (red), ICAM-1 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 7µg wt mTNF or 50µg of mCD13-AFR. Scale bar is 50µm.(D) Expression of ICAM-1, E-selectin and VEGF-R2 in B16Bl6 tumor 6h after treatment, by qPCR analysis of whole tumor RNA. Error bars are SEM. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by one-way ANOVA with Bonferroni's multiple comparison test.(E) Tumor growth and body weight change after daily p.l. injection of indicated (equimolar) doses of mCD13-AFR, non-targeted AFR (BcII10-AFR) or mCD13 VHH control (CD13-hIFNα2) in the B16Bl6 melanoma model. The line under the graph represents the treatment period. Tumor growth is shown as mean TSI, error bars are SEM (n = 5). ***, p < 0.001 by two-way ANOVA with Bonferroni's multiple comparison test.(F) B16Bl6 tumor growth after daily p.l. treatment with indicated doses of mCD13-AFR, Wortmannin (Wm), Birinapant (Bir) or combinations thereof. Wortmannin and Birinapant were given 1 hour before CD13-AFR. Tumor growth is shown as mean TSI + SEM (n = 4 for Wm and Bir, 5 for other groups). The line under the graph represents the treatment period. TSI of individual tumors of the indicated treatment groups at day 17 after tumor inoculation.


Figure 4 ( A ) Schematic representation of the CAR construct used , consisting of N - terminal anti - hCD70 VHH , hCD8α hinge and transmembrane ( TM ) domain , CD137 co - stimulatory domain and C - terminal CD3ζ activation domain . ( B , C ) SKOV3 tumor growth in NSG mice after i . v . injection of 6 x 106 hCD70 CAR T - cells on day 12 and p . l . treatment with 50µg mCD13 - AFR ( ↑ ) . Tumor growth is shown as mean TSI , error bars are SEM ( n = 5 ) ( B ) or as TSI of individual mice ( C ) . ( D ) Flow cytometric analysis of hCD45 + hCD3 + eGFP + CAR T - cells in SKOV3 tumors on day 18 after tumor inoculation . Mice were injected with 6 x 106 CAR T - cells on day 12 and treated with PBS or CD13 - AFR ( E ) B16Bl6 tumor growth and body weight change after daily p . l . injection of 30µg mCD8 - AFN , 50µg mCD13 - AFR or a combination thereof . Tumor growth is shown as mean TSI + SEM ( n = 6 ) . The line under the graph represents the treatment period . ( F ) TSI of individual tumors of the indicated treatment groups at day 19 after tumor inoculation .

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Figure 4(A) Schematic representation of the CAR construct used, consisting of N-terminal anti-hCD70 VHH, hCD8α hinge and transmembrane (TM) domain, CD137 co-stimulatory domain and C-terminal CD3ζ activation domain. (B,C) SKOV3 tumor growth in NSG mice after i.v. injection of 6 x 106 hCD70 CAR T-cells on day 12 and p.l. treatment with 50µg mCD13-AFR (↑). Tumor growth is shown as mean TSI, error bars are SEM (n = 5) (B) or as TSI of individual mice (C).(D) Flow cytometric analysis of hCD45+hCD3+eGFP+ CAR T-cells in SKOV3 tumors on day 18 after tumor inoculation. Mice were injected with 6 x 106 CAR T-cells on day 12 and treated with PBS or CD13-AFR(E) B16Bl6 tumor growth and body weight change after daily p.l. injection of 30µg mCD8-AFN, 50µg mCD13-AFR or a combination thereof. Tumor growth is shown as mean TSI + SEM (n = 6). The line under the graph represents the treatment period. (F) TSI of individual tumors of the indicated treatment groups at day 19 after tumor inoculation.


Figure 5 ( A , B ) B16Bl6 tumor growth after daily p . l . treatment with 7µg mTNF , 30µg hTNF alone or in combination with 10000 IU mIFN - γ in wt C57BL / 6 mice ( A ) or homozygous Flk1 dnIFN - γR1 transgenic mice ( B ) . The line under the graph represents the treatment period . Tumor growth is depicted as mean TSI , error bars are SEM ( n = 5 ) . ( C ) IL - 8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of sc hTNF mutant Y87F ( Y87F ) , hCD13 - AFR or wt hTNF , alone or in combination with 200ng / ml hIFN - γ , measured via ELISA . ( E ) Viability of B16Bl6 parental or mCD20 - expressing cells after 72h stimulation with wt mIFN - γ or del8 mutant fused to a BcII10 or mCD20 VHH , in the presence of 60ng / ml mTNF . Cell viability was measured via an ATP luminescence assay .

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Figure 5(A,B) B16Bl6 tumor growth after daily p.l. treatment with 7µg mTNF, 30µg hTNF alone or in combination with 10000 IU mIFN-γ in wt C57BL/6 mice (A) or homozygous Flk1 dnIFN-γR1 transgenic mice (B). The line under the graph represents the treatment period. Tumor growth is depicted as mean TSI, error bars are SEM (n = 5).(C) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of sc hTNF mutant Y87F (Y87F), hCD13-AFR or wt hTNF, alone or in combination with 200ng/ml hIFN-γ, measured via ELISA.(E) Viability of B16Bl6 parental or mCD20-expressing cells after 72h stimulation with wt mIFN-γ or del8 mutant fused to a BcII10 or mCD20 VHH, in the presence of 60ng/ml mTNF. Cell viability was measured via an ATP luminescence assay.


Figure 6 ( A , B ) Tumor growth , body weight change and rectal body temperature after daily p . l . treatment with mCD13 - AFR , mCD13 - AFN - II or combination thereof , in the B16Bl6 model in C57BL / 6 mice ( A ) or RL model in NSG mice ( B ) . Tumor growth is shown as mean TSI , error bars are SEM ( n = 5 ) ( upper panel ) , or as TSI of individual mice ( lower panel ) . ( C ) Immunohistochemical staining for PECAM - 1 ( red ) , cleaved Caspase - 3 ( green ) and DNA ( blue ) of B16Bl6 tumors after p . l . treatment with 50µg mCD13 - AFR and 23 . 8µg mCD13 - AFN - II . Scale bar is 100µm .

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

Figure 6(A,B) Tumor growth, body weight change and rectal body temperature after daily p.l. treatment with mCD13-AFR, mCD13-AFN-II or combination thereof, in the B16Bl6 model in C57BL/6 mice (A) or RL model in NSG mice (B). Tumor growth is shown as mean TSI, error bars are SEM (n = 5) (upper panel), or as TSI of individual mice (lower panel).(C) Immunohistochemical staining for PECAM-1 (red), cleaved Caspase-3 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 50µg mCD13-AFR and 23.8µg mCD13-AFN-II. Scale bar is 100µm.


Figure 1A : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . n = 6 for young and old mice , respectively . B : WT1 immunohistochemistry ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . n = 6 for young and old mice , respectively . C : TUNEL assay ( upper panels ) and quantification of TUNEL - positive tubular cells ( upper panel ) in glomeruli from young and aged mice . Panels are representative images of 6 young and old mice , respectively . Scale bar = 20 μm . D : Senescence - associated β - galactosidase staining ( upper panels ) and quantification of β - galactosidase - positive glomeruli ( lower panel ) in kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . Panels are representative images of 4 young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice . E : p21 / griffonia simplicifolia / nephrin coimmunostaining in kidneys from young and aged mice ( left panels ) and quantification ( right panel ) of p21 - positive endothelial cells per glomeruli . Original magnification X630 . Scale bar = 10 μm . Panels are representative images of 6 young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice . F : 53BP1 / CD34 co - immunostaining in kidneys from young and aged mice ( left panels ) and quantification ( right panels ) of 53BP1 foci per glomerular endothelial cell . Original magnification X630 . Scale bar = 10 μm . Panels are representative images of 6 young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice . I : PAI - 1 immunohistochemistry ( left panels ) and quantification of PAI - 1 - positive glomeruli ( right panel ) in kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . n = 5 for young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice .

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

Figure 1A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively. B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive tubular cells (upper panel) in glomeruli from young and aged mice. Panels are representative images of 6 young and old mice, respectively. Scale bar = 20 μm. D: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. Panels are representative images of 4 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.E: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys from young and aged mice (left panels) and quantification (right panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.F: 53BP1/CD34 co-immunostaining in kidneys from young and aged mice (left panels) and quantification (right panels) of 53BP1 foci per glomerular endothelial cell. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.I: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 5 for young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


Figure 2A : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 4 and n = 5 for RT + and RT - , respectively . B : WT1 immunohistochemistry ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 5 for RT + and RT - mice , respectively . C : Senescence - associated β - galactosidase staining ( upper panels ) and quantification of β - galactosidase - positive glomeruli ( lower panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 5 for RT + and RT - mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice . E : p21 / griffonia simplicifolia / nephrin coimmunostaining ( left panels ) and quantification of endothelial p21 - positive cells ( right panel ) in glomeruli of kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X630 . Scale bar = 10 μm . n = 4 for RT + and RT - mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice . F : 53BP1 / CD34 coimmunostaining ( left panels ) and quantification of glomerular endothelial cell 53BP1 foci ( right panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X630 . Scale bar = 10 μm . n = 4 for RT + and RT - mice , respectively . Arrows show 53BP1 foci . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice . H : PAI - 1 immunohistochemistry ( left panels ) and quantification of PAI - 1 - positive glomeruli ( right panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . n = 4 for RT + and RT - mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice .

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

Figure 2A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 5 for RT+ and RT-, respectively. B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively. C: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.E: p21/griffonia simplicifolia/nephrin coimmunostaining (left panels) and quantification of endothelial p21-positive cells (right panel) in glomeruli of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.F: 53BP1/CD34 coimmunostaining (left panels) and quantification of glomerular endothelial cell 53BP1 foci (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively. Arrows show 53BP1 foci. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.H: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. n = 4 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


Figure 3A : WT1 ( left upper panels ) and p21 ( left middle and lower panels ) immunostaining in 4 - , 12 - , 18 - and 24 - month - old mice . Quantification of WT1 - positive cells ( right upper panels ) , and glomerular and tubular p21 - postive cells ( right lower panels ) . Original magnification X400 . Scale bar = 20 μm . n = 4 for each group . Arrows show positive p21 nuclei , either in glomeruli or in tubules . B : WT1 ( left upper panels ) and p21 ( left middle and lower panels ) immunostaining in 4 - and 12 - month - old irradiated ( RT + ) and non - irradiated ( RT - ) mice . Quantification of WT1 - positive cells ( right upper panels ) , and glomerular and tubular p21 - postive cells ( right lower panels ) . Original magnification X400 . Scale bar = 20 μm . n = 4 for each group . Arrows show positive p21 nuclei , either in glomeruli or in tubules . Data information : Data are means ± SEM . Statistical analysis : ANOVA followed by the Tukey - Kramer test .

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

Figure 3A: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4-, 12-, 18- and 24-month-old mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. B: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4- and 12-month-old irradiated (RT+) and non-irradiated (RT-) mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test.


Figure 4A : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . B : WT1 immunostaining ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . C : PAI - 1 immunostaining ( upper panels ) and quantification of PAI - 1 - positive glomeruli ( lower panel ) in 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . Data information : Data are means ± SEM . Statistical analysis : t - student test : AP + versus AP - mice .

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

Figure 4A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. B: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. C: PAI-1 immunostaining (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. Data information: Data are means ± SEM. Statistical analysis: t-student test: AP+ versus AP- mice.


Figure 5A : PAI - 1 immunohistochemistry ( upper panels ) and quantification of PAI - 1 - positive glomeruli ( lower panel ) in kidneys of PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 4 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . B : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 12 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . C : TUNEL assay ( upper panels ) and quantification of TUNEL - positive glomeruli ( lower panel ) in kidneys from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . D : WT1 immunostaining ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in kidneys from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 4 and n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : PAI - 1 ∆ endo versus PAI - 1flox mice . G : p21 / griffonia simplicifolia / nephrin coimmunostaining in kidneys ( upper panels ) from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age and quantification ( lower panel ) of p21 - positive endothelial cells per glomeruli . Original magnification X630 . Scale bar = 10 μm . n = 4 and n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . H : 53BP1 / CD34 coimmunostaining ( upper panels ) and quantification of glomerular 53BP1 foci in glomerular endothelial cells from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X630 . Scale bar = 10 μm . n = 4 and n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . Arrows show 53BP1 positive foci . Data information : Data are means ± SEM . Statistical analysis : t - student test : PAI - 1 ∆ endo versus PAI - 1flox mice .

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

Figure 5A: PAI-1 immunohistochemistry (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in kidneys of PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 for PAI-1flox and PAI-1∆endo mice, respectively. B: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 12 for PAI-1flox and PAI-1∆endo mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive glomeruli (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. D: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.G: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys (upper panels) from PAI-1flox and PAI-1∆endo mice at 22 months of age and quantification (lower panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. H: 53BP1/CD34 coimmunostaining (upper panels) and quantification of glomerular 53BP1 foci in glomerular endothelial cells from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X630. Scale bar = 10 μm. n=4 and n=6 for PAI-1flox and PAI-1∆endo mice, respectively. Arrows show 53BP1 positive foci. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.


Figure 6C : Relative mRNA expression of senescence markers ( p16 and p21 ) and SASP molecules ( PAI - 1 , IL6 , IL8 ) in GEnC at early ( p9 ) or late ( p17 ) passage . n = 5 independent experiments . Data information : Data are means ± SEM . Statistical analysis : t - student testE : Immunofluorescence of paxilin ( green ) and phalloidin ( red ) in podocytes stimulated with control medium , supernatant ( SN ) from glomerular endothelial cells from early ( p9 ) or late ( p17 ) passage and treated either with vehicle or Tiplaxtinin , a PAI - 1 inhibitor ( upper panel ) . Quantification of focal adhesions ( lower panel ) was performed by counting the number of paxilin - positive spots in 30 cells from three independent experiments . Original magnification X1000 . Scale bar = 20 μm . Data information : Data are means ± SEM . Statistical analysis : ANOVA followed by the Tukey - Kramer

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6C: Relative mRNA expression of senescence markers (p16 and p21) and SASP molecules (PAI-1, IL6, IL8) in GEnC at early (p9) or late (p17) passage. n = 5 independent experiments. Data information: Data are means ± SEM. Statistical analysis: t-student testE: Immunofluorescence of paxilin (green) and phalloidin (red) in podocytes stimulated with control medium, supernatant (SN) from glomerular endothelial cells from early (p9) or late (p17) passage and treated either with vehicle or Tiplaxtinin, a PAI-1 inhibitor (upper panel). Quantification of focal adhesions (lower panel) was performed by counting the number of paxilin-positive spots in 30 cells from three independent experiments. Original magnification X1000. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test


Figure 7A : PAI - 1 staining ( left panel ) and quantification ( right panel ) in glomeruli of kidney biopsies from young ( < 40 years of age , n = 8 ) or old ( > 80 years of age , n = 10 ) kidney donors at time of transplantation ( M0 ) . Original magnification X400 . Scale bar = 20 μm . B : p16 staining ( left panel ) and quantification ( right panel ) in glomeruli of kidney biopsies from young ( < 40 years of age , n = 8 ) or old ( > 80 years of age , n = 10 ) kidney donors at time of transplantation ( M0 ) . The arrows point to the staining . Original magnification X400 . Scale bar = 20 μm . Data information : Data are means ± SEM . Statistical analysis : t - student testF : Quantification of the glomerulosclerosis score at time of ( M0 ) , or 12 months after ( M12 ) kidney transplantation from elderly donors in patients displaying a negative ( PAI - 1 - , n = 17 ) or positive ( PAI - 1 + , n = 13 ) glomerular PAI - 1 staining at time of transplantation ( M0 ) . Data information : Data are means ± SEM . Statistical analysis : ANOVA followed by the Tukey - Kramer testH : Urinary PAI - 1 measured by ELISA in a cohort of elderly patients with ( n = 48 ) or without ( n = 48 ) chronic kidney disease . Data information : Data are means ± SEM . Statistical analysis : t - student

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Figure 7A: PAI-1 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). Original magnification X400. Scale bar = 20 μm. B: p16 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). The arrows point to the staining. Original magnification X400. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: t-student testF: Quantification of the glomerulosclerosis score at time of (M0), or 12 months after (M12) kidney transplantation from elderly donors in patients displaying a negative (PAI-1-, n = 17) or positive (PAI-1+, n = 13) glomerular PAI-1 staining at time of transplantation (M0). Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer testH: Urinary PAI-1 measured by ELISA in a cohort of elderly patients with (n = 48) or without (n = 48) chronic kidney disease. Data information: Data are means ± SEM. Statistical analysis: t-student test


Figure 1Coomassie - stained SDS - PAGE gel showing purified protein from the predicted bacterial OTU constructs . Ub - PA activity - based probe assay for wild - type ( WT ) and catalytic Cys - to - Ala mutants ( CA ) . Strong , Cys - dependent reactivity is indicated with asterisks . Ub - KG ( TAMRA ) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations . Note that BurkOTU displays an increase in fluorescence polarization , indicative of noncovalent binding . Heatmap representation of DUB activity against the Ub - KG ( TAMRA ) substrate shown in G . , including the WT enzyme and Ala substitutions at the predicted catalytic Cys , general base His , or acidic position . Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants .

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Figure 1Coomassie-stained SDS-PAGE gel showing purified protein from the predicted bacterial OTU constructs.Ub-PA activity-based probe assay for wild-type (WT) and catalytic Cys-to-Ala mutants (CA). Strong, Cys-dependent reactivity is indicated with asterisks.Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations. Note that BurkOTU displays an increase in fluorescence polarization, indicative of noncovalent binding.Heatmap representation of DUB activity against the Ub-KG(TAMRA) substrate shown in G., including the WT enzyme and Ala substitutions at the predicted catalytic Cys, general base His, or acidic position. Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants.


Figure 2Ub / Ub - like specificity assay measuring activity of WT and inactive Cys - to - Ala wMelOTU toward the Ub - , ISG15 - , NEDD8 - , and SUMO1 - KG ( TAMRA ) substrates . Ub / Ub - like specificity assay measuring activity of WT and inactive Cys - to - Ala BurkOTU toward the Ub - , ISG15 - , NEDD8 - , and SUMO1 - KG ( TAMRA ) substrates . Note that the rise in fluorescence polarization signal , indicative of a noncovalent interaction , is specific to the Ub substrate . Heatmap representation of corrected OTU activities toward the Ub and Ub - like fluorescent substrates . In the reactions marked by an asterisk , an unusually high level of noise in fluorescence polarization signal was observed , likely a result of high OTU concentration . Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages . Reaction samples were quenched at the indicated timepoints , resolved by SDS - PAGE , and visualized by Coomassie staining . Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages . Reaction samples were quenched at the indicated timepoints , resolved by SDS - PAGE , and visualized by Coomassie staining . Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints .

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Figure 2Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala wMelOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala BurkOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Note that the rise in fluorescence polarization signal, indicative of a noncovalent interaction, is specific to the Ub substrate.Heatmap representation of corrected OTU activities toward the Ub and Ub-like fluorescent substrates. In the reactions marked by an asterisk, an unusually high level of noise in fluorescence polarization signal was observed, likely a result of high OTU concentration.Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints.


Figure 3Ub - KG ( TAMRA ) cleavage assay monitoring the effects of structure - guided wMelOTU mutations . These data were collected in parallel with those presented in Fig . 1G and the WT dataset is shown again for reference .

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Figure 3Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided wMelOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for reference.


Figure 4Ub - KG ( TAMRA ) cleavage assay monitoring the effects of structure - guided EschOTU mutations . These data were collected in parallel with those presented in Fig . 1G and the WT dataset is shown again for clarity .

Figure 4Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided EschOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for clarity.


Figure 5Ub - PA activity - based probe assay for WT vOTU and sequence - permutated vOTUP . Strong reactivity is indicated with asterisks . Ub - KG ( TAMRA ) cleavage assay monitored by fluorescence polarization for WT vOTU and sequence - permutated vOTUP .

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Figure 5Ub-PA activity-based probe assay for WT vOTU and sequence-permutated vOTUP. Strong reactivity is indicated with asterisks.Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization for WT vOTU and sequence-permutated vOTUP.


Figure 1About 25 μg of membrane ‐ enriched lysate from the mass spectrometry experiments was immunoblotted with anti ‐ PINK1 antibody . TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm , respectively .

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Figure 1About 25 μg of membrane‐enriched lysate from the mass spectrometry experiments was immunoblotted with anti‐PINK1 antibody. TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm, respectively.


Figure 3A - CConfirmation by mass spectrometry that Rab8A ( A ) , Rab8B ( B ) and Rab13 ( C ) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment . Flp ‐ In T ‐ Rex HEK293 cells expressing empty ‐ FLAG , WT PINK1 ‐ FLAG and KI ( D384A ) PINK1 ‐ FLAG were transfected either with HA ‐ Rab8A ( A ) , HA ‐ Rab8B ( B ) or HA ‐ Rab13 ( C ) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h . Whole ‐ cell lysates ( 10 mg ) were immunoprecipitated with anti ‐ HA agarose , resolved by SDS - PAGE and stained with colloidal Coomassie blue ( second panel ) . Coomassie ‐ stained bands migrating with expected molecular mass of HA ‐ Rabs were excised , in ‐ gel digested with trypsin and subjected to high ‐ performance liquid chromatography with LC ‐ MS / MS on an LTQ ‐ Orbitrap mass spectrometer . Upper panel shows the extracted ion chromatogram ( XIC ) analysis of Ser111 ‐ containing phosphopeptides ( 8A , NIEEHApSADVEK ; 8B , NIEEHApSSDVER ; 13 , SIKENApSAGVER ) with the combined signal intensity of the 2 + and 3 + forms of the peptide indicated on the y ‐ axis . Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1 ‐ FLAG ‐ expressing cells following CCCP stimulation . DCharacterisation of Rab8A , Rab8B and Rab13 phospho ‐ Ser111 antibodies . Flp ‐ In T ‐ Rex HEK293 cells expressing empty ‐ FLAG , WT PINK1 ‐ FLAG and KI ( D384A ) PINK1 ‐ FLAG were transfected with either WT or Ser111Ala ‐ mutant ( S111A ) HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 , induced with doxycycline and stimulated with 10 μM of CCCP for 3 h . Whole ‐ cell lysates ( 0 . 25 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rab8A , Rab8B or Rab13 phospho ‐ Ser111 antibodies . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls .

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Figure 3A-CConfirmation by mass spectrometry that Rab8A (A), Rab8B (B) and Rab13 (C) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected either with HA‐Rab8A (A), HA‐Rab8B (B) or HA‐Rab13 (C) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (10 mg) were immunoprecipitated with anti‐HA agarose, resolved by SDS-PAGE and stained with colloidal Coomassie blue (second panel). Coomassie‐stained bands migrating with expected molecular mass of HA‐Rabs were excised, in‐gel digested with trypsin and subjected to high‐performance liquid chromatography with LC‐MS/MS on an LTQ‐Orbitrap mass spectrometer. Upper panel shows the extracted ion chromatogram (XIC) analysis of Ser111‐containing phosphopeptides (8A, NIEEHApSADVEK; 8B, NIEEHApSSDVER; 13, SIKENApSAGVER) with the combined signal intensity of the 2+ and 3+ forms of the peptide indicated on the y‐axis. Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1‐FLAG‐expressing cells following CCCP stimulation.DCharacterisation of Rab8A, Rab8B and Rab13 phospho‐Ser111 antibodies. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected with either WT or Ser111Ala‐mutant (S111A) HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls.


Figure 4PINK1 is essential for CCCP ‐ mediated Rab8A Ser111 phosphorylation . WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala ( S111A ) mutant constructs of HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 . Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 ‐ 3xFLAG or KI ( D384A ) PINK1 ‐ 3xFLAG as indicated . After transfection for at least 24 h , cells were treated with DMSO as a vehicle control or CCCP for 20 h . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rab8A , Rab8B or Rab13 phospho ‐ Ser111 antibody . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls . For the lower panel , whole ‐ cell lysates ( 30 μg ) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls . Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent . WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h . Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 ‐ 3xFLAG or KI ( D384A ) PINK1 ‐ 3xFLAG as indicated for at least 24 h before CCCP treatment . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A pre ‐ bound with protein A agarose followed by immunoblot with Rab8A phospho ‐ Ser111 antibody . Part of the immunoprecipitates was used to immunoblot with anti ‐ total Rab8A antibody as loading controls .

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Figure 4PINK1 is essential for CCCP‐mediated Rab8A Ser111 phosphorylation. WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala (S111A) mutant constructs of HA‐Rab8A, HA‐Rab8B or HA‐Rab13. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated. After transfection for at least 24 h, cells were treated with DMSO as a vehicle control or CCCP for 20 h. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls.Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent. WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated for at least 24 h before CCCP treatment. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A pre‐bound with protein A agarose followed by immunoblot with Rab8A phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot with anti‐total Rab8A antibody as loading controls.


Figure 5Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts . Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control . Cells were incubated with DMSO or CCCP for 20 h , and whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho ‐ Ser111 antibody . Lysates ( 1 mg ) were also subjected to immunoprecipitation with polyclonal anti ‐ PINK1 antibody and immunoblotted with monoclonal PINK1 antibody . Equal loading of protein extracts was confirmed by GAPDH . Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs . MEFs were derived from PINK1 knockout embryos or wild ‐ type controls ( see Materials and Methods ) . Cells were incubated with DMSO or CCCP for 20 h , and whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho ‐ Ser111 antibody . Lysates ( 1 mg ) were also subjected to immunoprecipitation with a polyclonal anti ‐ mouse ‐ specific PINK1 antibody and immunoblotted with a different anti ‐ mouse ‐ specific PINK1 antibody . Equal loading of protein extracts was confirmed by GAPDH .

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Figure 5Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts. Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control. Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with polyclonal anti‐PINK1 antibody and immunoblotted with monoclonal PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs. MEFs were derived from PINK1 knockout embryos or wild‐type controls (see Materials and Methods). Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with a polyclonal anti‐mouse‐specific PINK1 antibody and immunoblotted with a different anti‐mouse‐specific PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.


Figure 6Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild ‐ type ( WT ) or Rab8A knockout ( KO ) HeLa cells were transfected with WT or Cys431Phe ( C341F ) ‐ mutant Parkin . After transfection for 24 h , cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h . Mitochondrial enriched extracts ( mitochondrial lysate ) were incubated with ubiquitin ‐ binding resins derived from his ‐ halo ‐ ubiquilin1 UBA domain tetramer ( UBAUBQLN1 ) . Captured ubiquitylated proteins were subject to immunoblotting with anti ‐ CISD1 and anti ‐ mitofusin 2 antibodies . Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls . Phospho ‐ Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti ‐ Rab8A antibody .

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Figure 6Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild‐type (WT) or Rab8A knockout (KO) HeLa cells were transfected with WT or Cys431Phe (C341F)‐mutant Parkin. After transfection for 24 h, cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h. Mitochondrial enriched extracts (mitochondrial lysate) were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBAUBQLN1). Captured ubiquitylated proteins were subject to immunoblotting with anti‐CISD1 and anti‐mitofusin 2 antibodies. Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls. Phospho‐Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti‐Rab8A antibody.


Figure 7In vitro phosphorylation analysis of Rab8A by PINK1 . WT or S111A ‐ mutant Rab8A ( 1 . 2 μg ) was incubated in the presence of MBP ‐ fused WT or KI ( D359A ) TcPINK1 ( 1 . 1 μg ) and Mg2 + ‐ [ γ ‐ 32P ] ATP for the indicated time . Samples were subjected to SDS - PAGE , and proteins were detected by colloidal Coomassie blue staining ( lower panel ) . The [ γ ‐ 32P ] incorporation to substrate was detected by autoradiography ( upper panel ) . Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [ γ ‐ 32P ] incorporation / mol of substrate . Ubiquitin was used as a positive control of the TcPINK1 substrate .

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Figure 7In vitro phosphorylation analysis of Rab8A by PINK1. WT or S111A‐mutant Rab8A (1.2 μg) was incubated in the presence of MBP‐fused WT or KI (D359A) TcPINK1 (1.1 μg) and Mg2+‐[γ‐32P] ATP for the indicated time. Samples were subjected to SDS-PAGE, and proteins were detected by colloidal Coomassie blue staining (lower panel). The [γ‐32P] incorporation to substrate was detected by autoradiography (upper panel). Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [γ‐32P] incorporation/mol of substrate. Ubiquitin was used as a positive control of the TcPINK1 substrate.


Figure 8Time ‐ course comparison of PINK1 ‐ mediated Rab8A , Rab8B and Rab13 Ser111 phosphorylation vs . Parkin Ser65 phosphorylation . Flp ‐ In T ‐ Rex HEK293 cells expressing WT PINK1 ‐ FLAG were transfected with either WT or Ser111Ala ‐ ( S111A ) mutant HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 , induced with doxycycline and stimulated with CCCP for the indicated time . In parallel , Flp ‐ In T ‐ Rex HEK293 cells expressing WT PINK1 ‐ FLAG were transfected with either WT or Ser65 Ala ( S65A ) ‐ mutant Parkin . Whole ‐ cell lysates ( 0 . 25 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with indicated phospho ‐ Ser111 antibodies . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls . For the lower panel , whole ‐ cell lysates ( 30 μg ) were immunoblotted with indicated antibodies . Time ‐ course comparison of endogenous PINK1 ‐ mediated Rab8A , Rab8B and Rab13 Ser111 phosphorylation vs . Parkin Ser65 phosphorylation . HeLa cells were transfected with either WT or S111A ‐ mutant HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 for at least 24 h before CCCP treatment for the indicated time . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with indicated phospho ‐ Ser111 antibodies . In parallel , HeLa cells were transfected with either WT or S65A ‐ mutant Parkin and whole ‐ cell lysates ( 30 μg ) were immunoblotted with indicated antibodies .

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Figure 8Time‐course comparison of PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser111Ala‐(S111A) mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with CCCP for the indicated time. In parallel, Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser65 Ala (S65A)‐mutant Parkin. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.Time‐course comparison of endogenous PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. HeLa cells were transfected with either WT or S111A‐mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13 for at least 24 h before CCCP treatment for the indicated time. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. In parallel, HeLa cells were transfected with either WT or S65A‐mutant Parkin and whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.


Figure 9Rabin8 ‐ catalysed mant ‐ GDP release from mant ‐ GDP loaded WT , S111A and S111E mutants of Rab8A . The Rab proteins ( 1 μM ) were incubated with 100 μM GDP in buffer ( 20 mM HEPES pH 7 . 5 , 50 mM NaCl , 1 mM MgCl2 , 2 mM DTE ) , and the reaction was started by the addition of 0 . 5 μM Rabin8 . The decrease in mant fluorescence was used as a measure of mantGDP release . Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells . Rab8A KO HeLa cells were transfected with wild ‐ type ( WT ) , S111E or S111A HA ‐ Rab8A . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rabin8 or anti ‐ HA antibody . Lysates were immunoblotted with Rabin8 or anti ‐ HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA ‐ Rab8A in extracts .

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Figure 9Rabin8‐catalysed mant‐GDP release from mant‐GDP loaded WT, S111A and S111E mutants of Rab8A. The Rab proteins (1 μM) were incubated with 100 μM GDP in buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM MgCl2, 2 mM DTE), and the reaction was started by the addition of 0.5 μM Rabin8. The decrease in mant fluorescence was used as a measure of mantGDP release.Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells. Rab8A KO HeLa cells were transfected with wild‐type (WT), S111E or S111A HA‐Rab8A. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rabin8 or anti‐HA antibody. Lysates were immunoblotted with Rabin8 or anti‐HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA‐Rab8A in extracts.


Figure 1B Mass spectrometry analysis of proteins co - purified with Ccq1HA . Shown is a volcano plot for proteins significantly enriched in Ccq1HA relative to untagged Ccq1 ( left panel : WT cells ; right panel : rik1 ∆ cells ) . Members of the CLRC complex and shelterin are highlighted in red and blue , respectively . Bottom panel displays proteins enriched ( log2 ≥ 5 or pval ≤ 0 . 01 ) ChIP - qPCR analysis of epitope - tagged shelterin components in WT and mutant strains as indicated ( negative control : untagged strain ) . Positions on x - axis denote distances relative to telomeric repeats ( telomere - proximal PCR primer ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ± SEMChIP - qPCR analysis of epitope - tagged shelterin components in WT and mutant strains as indicated ( negative control : untagged strain ) . Positions on x - axis denote distances relative to telomeric repeats ( telomere - proximal PCR primer ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ± SEMChIP - qPCR analysis of epitope - tagged shelterin components in WT and mutant strains as indicated ( negative control : untagged strain ) . Positions on x - axis denote distances relative to telomeric repeats ( telomere - proximal PCR primer ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ± SEMF ChIP analysis of Ccq1HA and FLAG - TetR - Clr4 * ( see scheme ) . Expression and tethering of FLAG - TetR - Clr4 * are controlled by thiamine / AHT addition ( red dots indicate induced / tethered FLAG - TetR - Clr4 * ; empty circles indicate non - induced / non - bound controls ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ±

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Figure 1B Mass spectrometry analysis of proteins co-purified with Ccq1HA. Shown is a volcano plot for proteins significantly enriched in Ccq1HA relative to untagged Ccq1 (left panel: WT cells; right panel: rik1∆ cells). Members of the CLRC complex and shelterin are highlighted in red and blue, respectively. Bottom panel displays proteins enriched (log2 ≥ 5 or pval ≤ 0.01)ChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEMChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEMChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEMF ChIP analysis of Ccq1HA and FLAG-TetR-Clr4* (see scheme). Expression and tethering of FLAG-TetR-Clr4* are controlled by thiamine/AHT addition (red dots indicate induced/tethered FLAG-TetR-Clr4*; empty circles indicate non-induced/non-bound controls) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM


Figure 2A Left : Schemes of CLRC and the shelterin complex . Middle and right : Scheme depicting the position of the ura4 + reporter gene and heterochromatin islands relative to telomeric repeats and TAS regions ; note that the minichromosomesome Ch16 does not contain TAS . B - C ChIP - qPCR analyses of H3K9me2 ( B ) and H3 ( C ) in WT and ccq1Δ cells ( n = 3 independent experiments . D Fold change of WT over ccq1Δ for H3K9me2 and H3 level Data information : In ( B , C ) , data are normalized to input and to the average of three euchromatic loci ( EC ) as internal contro and represented as mean ±

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Figure 2A Left: Schemes of CLRC and the shelterin complex. Middle and right: Scheme depicting the position of the ura4+ reporter gene and heterochromatin islands relative to telomeric repeats and TAS regions; note that the minichromosomesome Ch16 does not contain TAS. B-C ChIP-qPCR analyses of H3K9me2 (B) and H3 (C) in WT and ccq1Δ cells (n = 3 independent experiments. D Fold change of WT over ccq1Δ for H3K9me2 and H3 level Data information: In (B, C), data are normalized to input and to the average of three euchromatic loci (EC) as internal contro and represented as mean ± SEM


Figure 3ChIP - qPCR analyses of H3 in WT and mutants as indicated ( n = 3 independent experiments ) . Data information data are represented as mean ± SEChIP - qPCR analyses of H3 in WT and mutants as indicated ( n = 3 independent experiments ) . Data information data are represented as mean ± SEChIP - qPCR analyses of H3K9me2 in WT and mutants as indicated ( n = 3 independent experiments ) . Data information data are represented as mean ±

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Figure 3ChIP-qPCR analyses of H3 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SEChIP-qPCR analyses of H3 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SEChIP-qPCR analyses of H3K9me2 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SE


Figure 4A ChIP - seq reads mapped to TEL2R ( shown in reverse orientation for consistency )

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Figure 4A ChIP-seq reads mapped to TEL2R (shown in reverse orientation for consistency)


Figure 5A ChIP - qPCR analysis of H3 at endogenous TAS and a fragment spanning the TAS region from 115 - 905 bp ( relative to telomeric repeat ) inserted into the leu1 + locus ( see scheme ) . The TAS fragment was inserted into a strain that lacks endogenous TA Shown are ChIP analyses for WT ( left ) and ccq1 ∆ ( right ; note different the scale of the y - axis ) ( n = 9 - 10 independent experiments except for ectopic TAS in ccq1 ∆ strain where n = 3 ) Data information data are represented as mean ± SEB ChIP - qPCR analysis of H3 at reporter genes ( ura4 + and his3 + ) at various chromosomal locations ( see schemes ) in WT cells . TAS1 , TAS2 , and TAS3 correspond to position 116 bp , 2851 bp and 6291 bp ( relative to telomeric repeats ) , respectively ( n = 3 independent experiments ) . Data information data are represented as mean ±

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Figure 5A ChIP-qPCR analysis of H3 at endogenous TAS and a fragment spanning the TAS region from 115-905 bp (relative to telomeric repeat) inserted into the leu1+ locus (see scheme). The TAS fragment was inserted into a strain that lacks endogenous TA Shown are ChIP analyses for WT (left) and ccq1∆ (right; note different the scale of the y-axis) (n = 9-10 independent experiments except for ectopic TAS in ccq1∆ strain where n = 3) Data information data are represented as mean ± SEB ChIP-qPCR analysis of H3 at reporter genes (ura4+ and his3+) at various chromosomal locations (see schemes) in WT cells. TAS1, TAS2, and TAS3 correspond to position 116 bp, 2851 bp and 6291 bp (relative to telomeric repeats), respectively (n = 3 independent experiments). Data information data are represented as mean ± SE


Figure 6A , B RT - qPCR analysis of transcript levels of TERRA ( A ) and tlh1 + ( B ) in WT strain and mutants as indicated ( double mutants with ccq1Δ are indicated by blue dot ) . Data information : In ( A , B ) , data is represented as mean ± SEM from 3 independent experiments and shown relative to WT

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Figure 6A, B RT-qPCR analysis of transcript levels of TERRA (A) and tlh1+ (B) in WT strain and mutants as indicated (double mutants with ccq1Δ are indicated by blue dot). Data information: In (A, B), data is represented as mean ± SEM from 3 independent experiments and shown relative to WT leve


Figure 7A Genomic copy number of his3 + , ura4 + and TAS1 in indicated strains harboring the reporter genes tel1L : : his3 + and tel2L : : ura4 + . Cultures from individual WT and freshly generated knockout clones ( ccq1Δ , n = 16 ; clr4Δ , n = 5 ; ccq1Δ clr4Δ , n = 5 ) were pre - grown on selective media for several days and inoculated at day 0 to grow in liquid media with regular back - dilution ( every 24 hours , approximately 7 generations ) . Samples were taken at indicated harvest times , and relative copy numbers of genomic regions were assessed by qPCR ( normalization against intrachromosomal loci ) . Black and rainbow color lines indicate WT strains and individual ccq1 + deletion mutants ( clones ) , respectivelyB qPCR analysis as in ( A ) but with strains harboring the minichromosome Ch16 m23 : : ura4 + ( ccq1Δ , n = 14 ) . Since Ch16 is unstable in ccq1Δ cell Note that Ch16 does not harbor TAS

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Figure 7A Genomic copy number of his3+, ura4+ and TAS1 in indicated strains harboring the reporter genes tel1L::his3+ and tel2L::ura4+. Cultures from individual WT and freshly generated knockout clones (ccq1Δ, n = 16; clr4Δ, n = 5; ccq1Δ clr4Δ, n = 5) were pre-grown on selective media for several days and inoculated at day 0 to grow in liquid media with regular back-dilution (every 24 hours, approximately 7 generations). Samples were taken at indicated harvest times, and relative copy numbers of genomic regions were assessed by qPCR (normalization against intrachromosomal loci). Black and rainbow color lines indicate WT strains and individual ccq1+ deletion mutants (clones), respectivelyB qPCR analysis as in (A) but with strains harboring the minichromosome Ch16 m23::ura4+ (ccq1Δ, n = 14). Since Ch16 is unstable in ccq1Δ cell Note that Ch16 does not harbor TAS sequences


Figure 3Representative HDX - MS curves showing the protected regions in CD53 and CD2 . The HDX experiments were performed ( manually or with a LEAP automation system ) in triplicate . Error bars show the standard deviation . D Representative HDX - MS curves showing the protected regions in CD81 and CD19 . The HDX experiments were manually performed in triplicate . Error bars show the standard deviation .

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Figure 3Representative HDX-MS curves showing the protected regions in CD53 and CD2. The HDX experiments were performed (manually or with a LEAP automation system) in triplicate. Error bars show the standard deviation.D Representative HDX-MS curves showing the protected regions in CD81 and CD19. The HDX experiments were manually performed in triplicate. Error bars show the standard deviation.


Figure 5A CD81 knockout of 293T cells . The knockout cell line was generated by CRISPR - Cas9 . The immunoblots using CD81 antibody and actin antibody were conducted on the same film . B CD81 facilitates CD19 maturation . CD81 and CD19 ( 300 ng DNA ) were transfected into the CD81 - knockout 293T cells . Anti - FLAG immunoblot was performed for CD19 with a C - FLAG tag . The deglycosylation was performed by digesting the cell lysate with PNGase F or Endo H , following the manufacturers ' instructions . The deglycosylation pattern of CD19 species ( p : precursor , m and m ' : two mature forms ) differs because the glycosylation content of these species is differentC Trypsin digestion of CD19 . The digestion was performed at 37 ° C with intact cells . The mature CD19 forms ( m and m ' ) can be digested because they are surface expressed , whereas the intracellular precursor form ( p ) is protected from the digestion . E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19 . Chol : mutations at the cholesterol - binding site of CD81 . CD81 constructs and CD19 ( 300 ng DNA ) were co - transfected into the CD81 - knockout 293T cells . Top , Anti - FLAG immunoblot was performed for CD19 with a C - FLAG tag . In presence of wildtype CD81 ( wt ) , most CD19 is in the properly glycosylated mature form ( m ) , and the overglycosylated m ' form is not observed . With the EC1 mutants ( A4 in particular ) , however , m ' emerges and m shifts to high MW , indicating an alteration in the CD19 glycosylation . The level of intracellular p form is low with the wildtype CD81 or with the mutants . Bottom , Control experiment showing similar expression level of wild - type CD81 and CD81 mutants . Anti - V5 immunoblotting was conducted for the CD81 constructs with a C - V5 tag , and anti - actin immunoblotting was conducted on the same film .

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Figure 5A CD81 knockout of 293T cells. The knockout cell line was generated by CRISPR-Cas9. The immunoblots using CD81 antibody and actin antibody were conducted on the same film.B CD81 facilitates CD19 maturation. CD81 and CD19 (300 ng DNA) were transfected into the CD81-knockout 293T cells. Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. The deglycosylation was performed by digesting the cell lysate with PNGase F or Endo H, following the manufacturers' instructions. The deglycosylation pattern of CD19 species (p: precursor, m and m': two mature forms) differs because the glycosylation content of these species is differentC Trypsin digestion of CD19. The digestion was performed at 37 °C with intact cells. The mature CD19 forms (m and m') can be digested because they are surface expressed, whereas the intracellular precursor form (p) is protected from the digestion.E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19. Chol: mutations at the cholesterol-binding site of CD81. CD81 constructs and CD19 (300 ng DNA) were co-transfected into the CD81-knockout 293T cells. Top, Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. In presence of wildtype CD81 (wt), most CD19 is in the properly glycosylated mature form (m), and the overglycosylated m' form is not observed. With the EC1 mutants (A4 in particular), however, m' emerges and m shifts to high MW, indicating an alteration in the CD19 glycosylation. The level of intracellular p form is low with the wildtype CD81 or with the mutants. Bottom, Control experiment showing similar expression level of wild-type CD81 and CD81 mutants. Anti-V5 immunoblotting was conducted for the CD81 constructs with a C-V5 tag, and anti-actin immunoblotting was conducted on the same film.


Figure 6A Migration of naïve Cd53 - / - cultured primary pre - B cells to 100 ng / mL CXCL12 compared to WT littermate controls ( n = 5 mice per group ) over three independent experiments . Bars and error bars represent mean ± standard error of the mean . * * p < 0 . 01 by two - way ANOVA . B Migration of Cd53 - / - pre - B cells transduced with empty , control , or mutant constructs in response to CXCL12 gradient , 3 days after transduction ( n = 2 - 5 mice per group ) over two independent experiments . Bars and error bars represent mean ± standard error of the mean . * * p < 0 . 01 and * * * p < 0 . 001 by two - way ANOVA .

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Figure 6A Migration of naïve Cd53-/- cultured primary pre-B cells to 100 ng/mL CXCL12 compared to WT littermate controls (n = 5 mice per group) over three independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 by two-way ANOVA.B Migration of Cd53-/- pre-B cells transduced with empty, control, or mutant constructs in response to CXCL12 gradient, 3 days after transduction (n = 2-5 mice per group) over two independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 and ***p < 0.001 by two-way ANOVA.