Y-27632 Treatment has no Effect on Apoptosis and Necrosis in CSCs under Basal Conditions.

(A) No dose-dependent effect of Y-27632 on Caspase-3 activity under basal conditions. CSCs were treated with Y-27632 at the indicated concentrations for 48hrs. The representative phase contrast microscopy images are shown on the left. Scale bar = 100μm. Quantitative analysis using the Caspase-3 kit did not generate statistically significant differences between Control and Y-27632-treated groups (n = 15; ns: no significance). (B) Fluorescent microscope images of cell apoptosis and necrosis stained with Annexin V/PI. After treatment with 10μM Y-27632 for 48hrs, all cells (floating and adherent) were collected and stained with Annexin V-FITC/PI/Hoechst. The representative images show very few apoptotic cells (in green) and necrotic cells (in red) among many nuclei (in blue). Scale bar = 100μm. The corresponding flow data illustrates the necrotic cells (Q1), necrotic or late-apoptotic cells (Q2), early apoptotic cells (Q3), and normal cells (Q4), respectively. The distribution of subpopulations is shown in the bar graph on the right. No statistically significant differences for apoptotic and necrotic cells were found between Control and Treated groups (n = 3).