EMBO/SourceData huggingface dataset:
Figure 3qRT-PCR for INHBA relative to RPL27 using RNA from primary human fibroblasts transduced with a lentiviral vector allowing expression of INHBA in an inducible manner after treatment with DOX for 24 h (Fb Act, clones 1 and 2) or with empty vector (Fb EV) (N=3). Fb Act and Fb EV cultures were generated from lentivirally-transduced cells upon clonal expansion of resistant single cells.Western blot of conditioned media (CM) of transduced fibroblasts showing mature activin βA. Ponceau S staining of the membrane was used as a loading control.Chemotactic migration of SCC13 cells for 24 h in a transwell assay containing CM from Fb Act (clone 2) or Fb EV. N=4. Migration in one control culture was set to 1.Relative colony area of SCC13 cells upon culture in CM from Fb Act (clone 2) or EV. N=3.Representative immunofluorescence images of de-cellularized matrix from Fb Act (clone 2) and Fb EV stained for fibronectin or collagen I.Relative colony area of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=3.Migration of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=6. The experimental setup is shown schematically above the graph.Fb Act (clone 2) or Fb EV cells were cultured together in 2D with SCC13 cells. After 7 day of culture SCC13 cells were identified by K14 and the number of K14-positive colony area was quantified. N=6.Quantification of SCC13 tumor spheroid area in single hanging drop including CM from Fb EV or Fb Act (clone 2) (including 1% FBS), DMEM/1% FBS, or DMEM/1% FBS plus 20 ng/mL recombinant activin A. The area of the spheroid formed in one Fb EV sample was set to 1. N=9-10 hanging drops.Representative images and quantification of K14-stained areas from 3D cultures of SCC13 cells seeded on top of either Fb Act (clone 2) or Fb EV. N=3 independent cultures, n=3 histological sections.Representative pictures of tumors formed in NOD/SCID mice 6-7 weeks after intradermal injection of SCC13 cells with Fb Act (clone 2) or Fb EV. Tumor volume at different time points of tumor development (6-7 weeks after injection). N=5 tumors per group.
Figure 4mDia2 (Diap3) expression quantified by RNA sequencing of fibroblasts isolated from ear skin. N=3 pools of 3-6 mice.Primary mouse fibroblasts were treated with activin A or TGF-β1 or vehicle for 6h and analyzed by qRT-PCR (N=3) and Western blot for mDia2 (Diap3) and mDia2 expression, respectively. The GAPDH loading control is identical to the one in Fig 2C, since the same membrane was probed for mDia2, αSMA and GAPDH.Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX-inducible dominant-negative ActRIB mutant (dnActRIB) or an empty lentivirus (EV), treated with activin A for 2.5 h and analyzed by Western blot for mDia2 and GAPDH.Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act cells (clone 2) stained for mDia2 or K14 (green) (top), mDia2 (green) or MECA32/LYVE1 (red, bottom) and counterstained with Hoechst (blue).
Figure 5qRT-PCR for mDia2/DIAPH3 relative to RPL27 using RNA from normal human skin (N=11), SCCs (N=9) and BCCs (N=7).Representative skin/tumor sections from normal human skin, SCC and BCC patients stained for mDia2/DIAPH3. Note the strong mDia2/DIAPH3-positivity in the tumor stroma. Scale bars: 100 μm. D: Dermis, E: Epidermis: T: Tumor.Expression levels of mDia2/DIAPH3 in tumor and normal tissues based on TCGA cancer data (TCGA data version 20141017, http://firebrowse.org/). Gene expression levels were presented as normalized log2 RSEM (RNA-Seq by Expectation-Maximization) index from the database. BLCA: Bladder Urothelial Carcinoma (N = 408 & 19 tumor and normal tissue samples, respectively), BRCA: Breast Invasive Carcinoma (N = 1100 & 112), CESC: Cervical SCC (N = 306 & 3), ESCA: Esophageal SCC (N = 185 & 11), HNSC: Head-Neck SCC (N = 522 & 44), LIHC: Liver Hepatocellular Carcinoma (N = 373 & 47), LUSC: Lung SCC (N = 501 & 51), PAAD: Pancreatic Adenocarcinoma (N = 179 & 4), PRAD: Prostate Adenocarcinoma (N = 496 & 50), READ: Rectum Adenocarcinoma (N = 167 & 10), STES: Stomach and Esophageal Carcinoma (N = 600 & 46), UCEC: Uterine Corpus Endometrial Carcinoma (N = 546 & 35). Box plot shows relative mDia2/DIAPH3 expression in human tumor (red) and normal (blue) tissue. Boxes indicate the interquartile range, whiskers indicate data points within 1.5 times interquartile range, dots outside the whiskers indicate outliers, and the central line indicates median.Relative gene expression of mDia2/DIAPH3 and INHBA in the stroma of intrahepatic cholangiocarcinoma, breast and colorectal cancers vs. stroma of respective normal tissues based on datasets GSE45001 (D) (N = 10 normal tissue & 10 cancer samples, GSE9014 (E) (N = 4 & 13), and GSE35602 (F) (N = 6 & 53), and in CAFs from breast cancer samples vs. normal breast fibroblasts (GSE29270 dataset) (G) (N = 5 & 23). NS: Normal stroma, CS: Cancer stroma, NF: Normal fibroblasts, CAF: Cancer-associated fibroblasts). The expression values of the datasets were obtained using GEO2R tool in the GEO database.Kaplan-Meier survival curves based on datasets from GEO, EGA and TCGA, demonstrating that high expression of both mDia2/DIAPH3 and INHBA in patients with liver, stomach and breast cancer correlates with poor overall survival. Data and statistical analyses were collected from KMPlotter.
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).
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.
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.
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.
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.
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.
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.