Fig. 1

Genotoxic treatments cause distinct gene expression in human stromal cells with remarkable Zscan4 upregulation. a Representative images of human stromal cell (PSC27) senescence, developed 7–10 d after treatment by two different classes of anticancer agents. CTRL control, NDT non DNA-damaging treatment, DTX docetaxel, PTX paclitaxel, VCR vincristine, DT DNA-damaging treatment, BLEO bleomycin, MIT mitoxantrone, and RAD radiation. Upper panel, SA-β-Gal staining; lower panel, phase contrast. Yellow arrows, cytoplasmic vacuoles. Scale bars, 15 μm. b SA-β-Gal staining statistics of cells in the individual groups of (a). Data obtained from counting of at least 200 cells per independent experiment, and analyzed using Student’s t-test. c BrdU incorporation profiles of cells in the individual groups of (a), with representative images provided on the right (CTRL/DTX/BLEO shown for example; green, BrdU; blue, DAPI). At least 200 cells counted per independent assay. Scale bars, 15 µm. Data analyzed using Student’s t-test. d Quantification of DNA damage foci (DDF). The number of DDF per cell falls into each of the 0, 1–3, 4–10, and >10 counting categories. Right, representative images (CTRL/DTX/BLEO displayed for example; red, γH2AX; blue, DAPI). At least 200 cells counted per sample. Scale bars, 15 µm. Data analyzed using two-way ANOVA. e GO analysis (cellular component, top 100 genes) of cells treated by DT and NDT agents, respectively. Color annotations shown on the right of Panther pie charts. f Heatmap depiction of top 30 human genes significantly upregulated in stromal cells after treatment by DT agents, with expression profiles generated by NDT agents shown side-by-side for parallel comparison. Numeric values in the bracket per gene refer to the expression fold change of the DT groups against the control. g Immunoblot analysis of the expression or activation of proteins including Zscan4, 53BP1, p38, p16, p21, and a subset of canonical SASP factors. Unless noted, data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, ^ P > 0.05