Pretreatment with Y-26732 Decreased Caspase-3 Activity in CSCs Induced by Dox.

(A) Fluorescent microscope images of apoptotic/necrotic cells stained with Annexin V/PI kit. CSCs were pretreated with 10μM Y-27632 for 48hrs followed by 48hrs exposure to 0.6μM Dox. Annexin V (a1 to a4, in green), Hoechst (a5 to a8, in blue), and PI (a9 to a12, in red) fluorescent images show the reduced apoptotic (a4) and necrotic (a12) cells compared with Controls (a2 and a10), respectively. Scale bar = 100μm. (B) Effect of pretreatment with Y-27632 on apoptosis induced by Dox. CSCs were pretreated with various concentrations (0.0, 0.1, 1.0, & 10.0μM) of Y-27632 for 48hrs followed by 48hrs exposure to 0.6μM Dox. The representative phase contrast microscope images were taken at the end of each treatment. Control (b1) refers to “no Y-27632, no Dox” and the rest of the images (b2 to b5) indicate the tested concentrations of Y-27632 plus 0.6μM Dox. Scale bar = 100μm. The Caspase-3 kit was used to determine the Caspase-3 activity in the plate reader and the means of the fold changes are shown in the bar graphs in the upper right hand corner (n = 18; ns: no significance, *: p