Research Paper Volume 16, Issue 21 pp 13252—13270

Figure 1. Characterization of etoposide-induced senescence in human chondrocytes at day 7. (A) Schematic workflow of etoposide (ETO)-induced senescence in osteoarthritic chondrocytes. (B) Level of BrdU incorporation in non-treated (NT) and ETO-treated chondrocytes (n = 3). (C) Percentage of proliferation in NT and ETO chondrocytes (n = 3). (D) Relative expression of Cyclin-Dependent Kinase Inhibitors in NT and ETO chondrocytes by RT-qPCR (n = 3–7). (E) Representative pictures of SA-β-Galactosidase (Gal) staining in NT and ETO chondrocytes (left panel; bars: 100 µm). Percentage of SA-β-Gal-positive cells (n = 7) and SA-β-Gal activity quantified by fluorometry (n = 3) (right panel). (F) Representative staining of actin stress fibers (in red) and nuclei (in blue) with Phalloidin and DAPI, respectively (left panel; bars: 100 µm). Quantification of the cell surface (n = 12) and corrected total cell fluorescence (CTCF) (n = 9) (right panels). (G) Representative pictures of γH2AX-positive foci (red spot) in DAPI stained nuclei (blue) (left panel; bars: 50 µm). Percentage of cells with γH2AX-positive nuclei (n = 8) and quantification of nucleus surface (n = 7) (right panel). (H) Protein secretion in supernatants of NT and ETO chondrocytes quantified by ELISA (n = 6). Data are shown as mean ± SEM. Statistical analysis used the Mann-Whitney test (B, D: pairwise comparisons, E: left panel, F, G, H) or Wilcoxon signed rank test (C, E: right panel). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.