Fig 2: Avn C reduces H2O2-induced oxidative stress and DNA double-strand damage in 2DD fibroblasts. Normal human skin fibroblasts (2DD) were pre-treated with DMSO (0) or Avn C (100 μM and 200 μM) for 48 h followed by 1 h exposure to 200 μM H2O2. (A) Representative images from cells stained with MitoTracker Orange dye. MitoTracker Orange reacts with free radicals inside cells to generate orange fluorescence. Cell chromatin is counterstained with DAPI (blue) to indicate nuclear location. Scale bar: 50 μm. (B) qRT-PCR on cDNA libraries was performed for glutathione synthetase (GSS), heme oxygenase 1 (HMOX1), superoxide dismutase 1 (SOD1), glutathione superoxidase 1 (GPx1) and catalase (CAT) gene transcripts. Data are represented as the fold change against control. (C) Western blot analysis of γ-H2AX expression. Data are presented as a ratio of γ-H2AX to β-actin. Representative blot images are shown. All graphs indicate mean values from three biological replicates. Error bars represent standard deviations. *p value < 0.05, **p value < 0.01 vs H2O2 treatment alone.

Fig 3: Downregulation of SEPHS1 in OA cartilage leads to oxidative stress-induced cellular senescence in chondrocytes.a Relative mRNA expression level of Sephs1 in primary cultured chondrocytes isolated from Sephs1fl/fl or Sephs1fl/fl; Col2a1-Cre mice (n = 4). b Western blot analysis of selenoproteins in primary cultured chondrocytes isolated from Sephs1fl/fl (n = 4) and Sephs1fl/fl; Col2a1-Cre mice (n = 3). c Quantification of protein levels in (b) (n = 4, 3 respectively). d, e Fluorescence-activated cell sorting (FACS) analysis of d CM-H2DCFDA and e DHE fluorescence in primary cultured chondrocytes isolated from Sephs1fl/fl and Sephs1fl/fl; Col2a1-Cre mice. f, g Immunofluorescence staining and quantification of f CM-H2DCFDA (n = 6) and g DHE (n = 4) fluorescence in chondrocytes transfected with negative control siRNA or siRNA targeting Sephs1. h GSEA of ‘Cellular senescence’ and ‘Oxidative stress-induced senescence’ gene sets in chondrocytes transfected with negative control siRNA or siRNA targeting Sephs1. i Immunofluorescence staining of γ-H2AX and quantification of γ-H2AX positivity in primary cultured chondrocytes isolated from Sephs1fl/fl and Sephs1fl/fl; Col2a1-Cre mice (n = 4). j SA-β-Gal staining and quantification of SA-β-Gal positivity in primary cultured chondrocytes isolated from Sephs1fl/fl and Sephs1fl/fl; Col2a1-Cre mice (n = 6). k, l Quantification of k immunofluorescence positivity of γ-H2AX and l SA-β-Gal positivity in primary cultured chondrocytes transfected with negative control siRNA or siRNA targeting Sephs1 followed by NAC treatment at the indicated doses (n = 4). m Relative mRNA expression of SASP factors in chondrocytes transfected with negative control siRNA or siRNA targeting Sephs1 (n = 6). n GSEA of the ‘Upregulated genes in OA’ gene set in chondrocytes transfected with negative control siRNA or siRNA targeting Sephs1. Scale bars: f, i, k 25 μm, j, l 50 μm. a, c, f, g, i–m Data represent means ± s.e.m. P values are from two-tailed t test (a, c, f, g, i, j, m) or two-way ANOVA followed by Dunnett’s post-hoc test (k, l). For GSEA plots in h, n, enrichment plots are displayed with the determined nominal P value and normalized enrichment score (NES). Unprocessed immunoblot images are provided in Supplementary Fig. 10.

Fig 4: The inhibition of IRE1-alpha/XBP1 axis increases the cytotoxic effect of PARP inhibition also in PEL cells. BCBL1 cells were pre-treated with 4μ8C (40 μM) for 1 h and subsequently treated with AZD2461 (AZD) (40 μM). (A) After 24 h, the cell viability was measured by a Trypan Blue exclusion assay. The histograms represent the mean plus S.D. of live cells as percent of untreated control cells. (B, C) Protein expression level of XBP1s, PARP and γ-H2AX was evaluated by Western blot analysis. β-Actin was used as loading control and one representative experiment is shown. The histograms represent the densitometric analysis of the ratio of specific protein and β-Actin of three different experiments. Data are represented as the mean plus S.D. p value: * p < 0.05; ** p < 0.001; *** p < 0.0001.

Fig 5: VPA and TSA increase the DNA damage induced by AZD2461 PARP1 inhibitor. PaCa44 and Panc1 cells were pre-treated with VPA (10 mM) and TSA (500 nM) for 1 h and then treated or not with AZD (10 μM). (A,B) phosphorylation of H2AX was evaluated by Western blot analysis after 36 h of treatments; actin was used by loading control. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio between the protein and the appropriate control. * p-value < 0.05. (C,D) γH2AX foci (red) were assessed by IFA. DAPI (blue) was used for nuclear staining. Images were captured under ApoTome microscope at ×40 magnification. Bars = 10 μm. The number of foci/cells, calculated as mean plus S.D. from three independent experiments, are represented by the histograms shown on the right side of the figures C (PaCa44) and D (Panc1). * p-value < 0.05.