Figure 4.
Pro-apoptotic effects of combined OC-LP treatment in HER2-positive BC cells. (A) Flow cytometry analysis for OC combined with LP treatment in BT-474 and SK-BR-3 BC cells. Cells were plated at a density of 5 × 106 cells/100 mm culture plates, allowed to attach overnight. Afterwards, cells were incubated in the respective control, OC, LP, or LP-OC combination in RPMI-1640 medium containing 40 ng/mL HGF and EGF for 24 h. At the end of the experiment, cells in each treatment group were trypsinized, washed then resuspended in ice-cold 1X Annexin V binding buffer. Afterwards, cells treated as described earlier. In the dot plot of double variable flow cytometry, LL quadrant (FITC−/PI−) shows living cells; LR quadrant (FITC+/PI−) represents early apoptotic cells; UR quadrant (FITC+/PI+) stands for late apoptotic cells and UL quadrant (FITC+/PI+) stands for necrotic cells. (B) Western blot analysis of cleaved caspase 3 and cleaved PARP performed after 24 h incubation. Cells analyzed to examine cell death by measuring cleaved caspase 3 and cleaved PARP detected by Western blotting. In all the above experiments, whole cell lysates were prepared for subsequent separation by polyacrylamide gel electrophoresis followed by Western blot analysis. Imaging and analysis performed as described earlier. * p < 0.05, *** p < 0.001 as compared with either individual OC or LP treatment or vehicle treatment.