Fig. 4: Combined GSK3β and PARP inhibition increases DNA damage and mitotic aberrancies. | Cell Death & Disease

Fig. 4: Combined GSK3β and PARP inhibition increases DNA damage and mitotic aberrancies.

From: Glycogen synthase kinase 3β inhibition synergizes with PARP inhibitors through the induction of homologous recombination deficiency in colorectal cancer

Fig. 4

A Western blot analysis of indicated proteins in HCT-15 cells (parent) or corresponding GSK3β-depleted single clone (KO1 and KO2) cells treated with 5 μM simmiparib (SP) for 48 h. B Western blot analysis of indicates proteins in HCT-15 cells treated with 5 μM simmiparib, GSK3i (10 μM CHIR99021 HCl or 5 μM LY2090314), or a combination for 48 h. SP, simmiparib; CHIR, CHIR99021 HCl; LY, LY2090314. C and D Representative images of p-RPA32 (S33) and γ-H2AX foci in HCT-15 (parent) and their GSK3β-depleted single clone (KO1 and KO2) cells treated with 5 μM simmiparib (C) or HCT-15 cells following treatment with 5 μM simmiparib, GSK3i (5 μM LY2090314), or a combination for 48 h (D). Nuclei were stained with DAPI. Scale bar: 2 μm. Cells that contained five or more p-RPA32 or γ-H2AX foci/nucleus were considered as positive cells. At least 50 cells were analyzed for each experiment and condition. All data are expressed as mean ± SD from three independent experiments. (C: ***p < 0.001, t test; D: ***p < 0.001, one-way ANOVA). SP, simmiparib; CHIR, CHIR99021 HCl; LY, LY2090314. E The combination of GSK3i and PARPi increases anaphase bridge-positive cells. HCT-15 cells were treated with 5 μM simmiparib (SP), 5 μM LY2090314 (LY), or a combination for 48 h. Cells were examined by DAPI-staining and microscopy for chromatin bridges. Representative images (upper panel; scale bar: 2 μm) and percentages of anaphase bridge-positive cells are shown (lower panel; *p < 0.05, **p < 0.01, one-way ANOVA). Anaphase cells (Control: 45 cells; SP: 44 cells; LY: 47 cells; SP + LY: 52 cells) from five independent experiments. Data are expressed as mean ± stand error of mean (SEM). F The combination of GSK3i and PARPi impairs mitotic spindles. HCT-15 cells were immunostained with α-tubulin (green) for mitotic spindles and pericentrin (red) for centrosomes after treated with 5 μM simmiparib (SP), 5 μM LY2090314 (LY), or a combination for 48 h. Nuclei were stained with DAPI. Representative images (upper panel; scale bar: 2 μm) and percentages of abnormal spindles are shown (lower panel; ***p < 0.001, one-way ANOVA). At least 50 cells were analyzed for each experiment and condition. Data are expressed as mean ± SD from three independent experiments.

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