4.7. Western Blot Analysis

A2780-SP cells were plated in Corning ultra-low attachment 6-well plates at a density of 4 × 10⁵ viable cells per well and grown in CSC medium, followed by starvation of the sphere cells with NBM. After 16 h, the cells in NBM were treated with the compounds. After 1 h, the cells in the CSC medium were treated with the compound. After 30 min, the cells were harvested to extract the protein. Cell lysates were separated by SDS-PAGE gel and transferred to PVDF membranes for Western blot analysis. After blocking with 5% skim milk, the membranes were initially incubated with primary antibodies in blocking buffer overnight at 4 °C, followed by HRP-conjugated secondary antibodies for 2 h at RT. The following primary antibodies were used: anti-phospho-AKT (Ser473) (#3787S), anti-AKT (#9272S), anti-phospho-ERK (Thr202/Tyr204) (#9101S), anti-ERK (#9102S), anti-phospho-p38 (#9211S), anti-p38 (#9212S), anti-OCT3/4 (sc-8682), anti-NANOG (#3580S), anti-SOX2 (#3579S), anti-ALDH1 (#611194), anti-CD133 (ab19898) and anti-GAPDH (sc-47724), anti-MCL1 (#4572S), anti-BCL2 (sc-7382), anti-SURVIVIN (#2803S), anti-cleaved Caspase3 (#9661S), anti-Caspase3 (#9665S), anti-cleaved PARP (#5625S), and anti-PARP (#9542S). The secondary antibodies used were goat anti-mouse IgG-HRP (Bioss, Woburn, MA, USA) and goat anti-rabbit IgG-HRP (Bioss, San Diego, CA, USA). Signals were developed with enhanced chemiluminescence HRP substrate (Bio-Rad, Hercules, CA, USA) and detected using LAS-3000 mini (Fuji film). The signal intensities were calculated with ImageJ software (NIH Image, USA).