Fig 2: Inactivation of YAP is required for talazoparib to promote cellular senescence in HepG2 cells. HepG2 cells were treated with DMSO (0.02%, w/v) or talazoparib at 5 µmol/L concentrations for 24 h. A, Western blot analyses of YAP expression. B, Immunofluorescence staining for YAP expression. Scale bar, 50 µm. HepG2 cells were stably transfected with YAP plasmid construction for 6 h and then treated with the 5 µmol/L concentration of talazoparib for 24 h. C, Western blot analysis of the transfection efficiency. D, Cell Counting Kit-8 was assayed for detecting the cell viability. E, Immunoblot analysis of p16, p21, Hmga1 and ?-H2AX was performed. F, The expression of p16, p21 and Hmga1 was assessed by immunofluorescence. Scale bar, 50 µm. Data are expressed as mean ± SD (n = 3); ***P < .001 vs DMSO. # P < .05 vs talazoparib

Fig 3: DAU combined with ATR inhibitors induces DNA damage in a MYC-dependent manner. A, Immunoblots of the indicated proteins in SW480, HCT116, and RKO cells treated for 24 hours with DAU alone or together with BAY-1895344. B, Phosphorylation of H2AX in HCT116, SW480, and RKO cells treated with DAU and BAY-1895344 alone or together were revealed by immunostaining. Phospho-H2AX (14) and DAPI (blue). Quantified data are shown in C. #, P < 0.0001; significantly different from vehicle-treated group. D, Immunoblots of the indicated proteins in SW480, HCT116, and RKO cells treated with DAU and BAY-1895344 alone or together for 24 hours. E and F, Immunoblots of the indicated proteins in control and MYC knockdown SW480 (E) and HCT116 (F) cells treated with DAU alone or together with BAY-1895344, VE-822, or AZD6738 for 24 hours. G, Phosphorylation of H2AX in ARPE-19-MYC (DOX+) and ARPE-19-MYC (DOX-) cells treated with 20 µmol/L DAU and 50 nmol/L BAY-1895344 alone or together for 16 hours. Quantified data are shown in H. #, P < 0.0001. I, Immunoblots of the indicated proteins in ARPE-19-MYC (DOX+) and ARPE-19-MYC (DOX-) cells treated with DAU and BAY-1895344 with or without 50 µmol/L cytidine (cyti), uridine (urid), guanosine (guan), or adenosine (aden). Graphic data are means ± SEM. For Western blotting, one of three to five similar experiments is shown. Scale bar, 20 µm. Ctrl, control; DOX, doxycycline.

Fig 4: scRNA-seq identifies a reduction in H2ax expression in beclin 1+/- mouse corneal epithelial cells. (A–D) Immunofluorescence staining with H2AX antibody in a frozen section of mouse limbal and corneal epithelium. In wild-type mice, H2AX is preferentially expressed in the corneal epithelium (B) compared with limbal epithelium (A). H2AX expression is significantly decreased in the corneal epithelium from beclin 1+/- mice (D). n = 4. Scale bar: 50 µm. (E, F) Immunofluorescence staining with H2AX antibody in a frozen section of human limbal (F) and corneal (E) epithelium. H2AX was primarily restricted to the corneal epithelium. Scale bar: 20 µm. Dotted lines mark the basement membrane. n = 3.

Fig 5: ZC-22 inhibits CDK4/6 and PARP to induce cell-cycle arrest, DNA damage, and apoptosis in breast and ovarian cancer cells. (A,B) MDA-MB-231 cells were treated with 2 µM LY2835219 (LY-2) or ZC-22. OVCAR5 cells were treated with 0.5 µM LY-2 or ZC-22, for 24 h. The levels of CDK4/6 and p-Rb/Rb were determined by Western blot (A). Cell-cycle distribution was analyzed by flow cytometry (B). Values are means ± SD, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001 for comparisons with the DMSO-treated group at each phase in an unpaired t-test. (C) Western blot analysis of PARP, PAR, ?H2A.X, and H2A.X in MDA-MB-231 cells treated with 2 µM Olaparib (OLA) or ZC-22 for 48 h (Left), or OVCAR5 cells treated with 0.5 µM OLA or ZC-22 for 48 h (Right). (D,E) Representative pictures (left) and quantification of percentage (right) of ?H2A.X-positive cells in MDA-MB-231 cells treated with 2 µM OLA or ZC-22 for 24 h (D), or OVCAR5 cells treated with 0.5 µM OLA or ZC-22 for 24 h (E). Values are means ± SD, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001 for comparisons with the DMSO-treated group. p value was calculated using an unpaired t-test. (F,G) Representative pictures (F) and quantification of percentage (G) of apoptotic cells in MDA-MB-231 cells treated with 2 µM LY-2, OLA, or ZC-22 for 48 h or in OVCAR5 cells treated with 0.5 µM LY-2, OLA or ZC-22 for 48 h. Values are means ± SD, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the DMSO-treated group. p value was calculated using an unpaired t-test.