Fig. 5: HR488B induces apoptosis in CRC cells via mitochondrial dysfunction, ROS accumulation, and DNA damage. | Cell Death & Disease

Fig. 5: HR488B induces apoptosis in CRC cells via mitochondrial dysfunction, ROS accumulation, and DNA damage.

From: Targeting the E2F1/Rb/HDAC1 axis with the small molecule HR488B effectively inhibits colorectal cancer growth

Fig. 5

a Apoptosis of HCT116 and HT29 cells induced by DMSO or HR488B (0.2, 0.5, and 1 μM) treatment for 48 h were examined by using Annexin V-FITC/PI double-staining analysis. b The statistical result of (a). c HCT116 and HT29 cells were incubated with DMSO or HR488B (0.2, 0.5, and 1 μM) for 24 h. PARP, cl-PARP, caspase 3, cl-caspase 3, Bax, Bcl-2, and Cyto C protein levels were examined by Western blot analysis, and β-actin was detected as the endogenous loading control, accordingly. d The statistical result of (c). e HCT116 and HT29 cells were treated with DMSO or 0.5 and 1 µM of HR488B for 24 h. The treated cells stained with JC-1 were pictured by fluorescence microscope to estimate the alteration in MMP (Scale bar, 100 μm). f HCT116 and HT29 cells were treated with DMSO or HR488B (0.2, 0.5, and 1 μM) for 24 h. Representative flow cytometry histograms displaying levels of fluorescent DCFH-DA in cells were presented. g Statistical analysis of the percentage of ROS generation (f). h HCT116 and HT29 cells were pretreated with or without NAC (5 mM) for 2 h, then treated with HR488B (1 μM) and NAC (5 mM) alone or in combination for 24 h. Intracellular ROS was measured by flow cytometry after 10 μM DCFH-DA staining. i Statistical analysis of the percentage of ROS generation (h). j HCT116 and HT29 cells were treated with DMSO or HR488B (0.2, 0.5, and 1 μM) for 24 h on DNA damage detected by alkaline comet assay (Scale bar, 50 μm). k HCT116 and HT29 cells were treated with DMSO or HR488B (0.2, 0.5, and 1 μM) for 24 h, and the expression of γ-H2AX was analyzed by immunofluorescence. Green represents the cells stained with anti-γ-H2AX antibody, and blue represents the nuclei stained with DAPI (Scale bar, 25 μm). l HCT116 and HT29 cells were pretreated with or without NAC (5 mM) for 2 h, then treated with HR488B (1 μM) and NAC (5 mM) alone or in combination for 24 h. PARP, cl-PARP, Bax, Bcl-2, E2F1, and γ-H2AX protein expression were evaluated by Western blot analysis, and β-actin was detected as the endogenous loading control, accordingly. m The statistical result of (l). All data are shown as mean ± SD, n = 3, two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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