Fig. 3

Zscan4 overexpression does not change stress-induced cell phenotypes but alters SASP expression and DDR signaling. a Statistics of DDR foci in PSC27 stable sublines established via lentiviral infection and treated by bleomycin (50 µg/ml). The number of cells displaying each category of DDR foci was counted and converted into percentage per cell population. Right, representative images (red, γH2AX; blue, DAPI). At least 200 cells counted per sample. Scale bars, 10 µm. Data analyzed by two-way ANOVA. b DNA synthesis evaluation by BrdU incorporation of cell sublines assayed in a, with BrdU-positive cells calculated in percentage. Right, representative images (green, BrdU; blue, DAPI). At least 200 cells counted per sample. Scale bars, 15 µm. Data analyzed by Student’s t-test. c Cellular senescence assessment by SA-β-Gal staining of above cell sublines, with positively stained cells shown in percentage. Right, representative images. At least 200 cells counted per sample. Scale bars, 20 µm. Data analyzed by Student’s t-test. d Transcript expression of canonical SASP factors in stromal sublines treated in a, with values normalized to the vector control per factor. Data analyzed by Student’s t-test. e Left, immunoblot analysis of inducible expression of Zscan4 and activation of DDR factors including H2AX, ATM, Kap1, Chk2, and p53 in primary PSC27 cells treated by chemotherapeutic agents. DXR, doxorubicin, a supplementary genotoxic control; Right, cells were lentivirally transduced with scramble-shRNA (SCR-shRNA) or gene-specific shRNA for Zscan4 (Zscan4-shRNA), and treated by bleomycin. Cell lysates were collected at the individual time points post treatment for immunoblotting against Zscan4 expression and DDR factor activation. Data in all bar plots are shown as mean ± SD and representative of 3 representative biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ^P > 0.05