Figure 7

(A) Western immunoblotting of whole-cell lysates of treated MCF-7 and T47D cells for the expression of Bad. MCF-7 and T47D cells were treated with ATRA and VN/14-1 for 6 days and then cell lysates were electrophoresed using 15% SDS–PAGE and subjected to Western blotting. Lane 1: control; lanes 2–4: ATRA (1, 5 and 10 μ M); and lanes 5–7: VN/14-1 (1 5 and 10 μ M). Numbers below the blot show fold increase in expression of the protein as analysed by ImageQuant densitometry analysis. Membranes were stripped and probed for β-actin to verify equal protein loading. Primary antibody (Cell Signaling Technology) 1 : 1000 in 5% BSA TBST overnight at 4°C and secondary antibody (anti-rabbit) 1 : 2000 for 1 h at RT. This experiment was repeated twice with similar results. (B) Western blot showing activation of caspase-9 in T47D cells after treatment with ATRA or VN/14-1. T47D cells were treated with ATRA or VN/14-1 for 6 days and whole-cell lysates were electrophoresed using 10% SDS–PAGE and subjected to Western immunoblotting for caspase-9. Treatment with ATRA and VN/14-1 cleaved procaspase-9 to its active form. Lane 1: control; lanes 2–4: ATRA (1, 5 and 10 μ M); and lanes 5–7: VN/14-1 (1, 5 and 10 μ M). Numbers below the blot show fold increase in the active form of caspase-9 as compared with control. Membrane was stripped and probed for β-actin to verify equal amount of protein loading. The primary and secondary antibodies and their dilutions are as follows: primary antibody (Cell Signaling Technology), 1 : 1000 in 10% milk TBST overnight at 4°C and secondary antibody (anti-rabbit) 1 : 2000 in 10% milk TBST for 1 h at RT. Densitometry analysis was performed by ImageQuant software. This experiment was repeated twice with similar results. (C) Western immunoblotting of whole-cell lysates of treated MCF-7 and T47D cells for the expression of full-length and cleaved PARP. MCF-7 cells were treated with ATRA and RAMBAs for 6 days and 4-HPR for 4 days and then cell lysates were electrophoresed using 10% SDS–PAGE and subjected to Western blotting. Lane 1: control; lanes 2–4: VN/14-1 (1, 5 and 10 μ M); lane 5: ATRA (5 μ M); lane 6: VN/50-1 (5 μ M); lane 7: VN/66-1 (5 μ M); lane 8: VN/69-1(5 μ M); and lane 9: 4-HPR (5 μ M). T47D cells were treated with RAMBAs (VN/50-1 and VN/14-1) for 6 days and then cell lysates were electrophoresed using 10% SDS–PAGE and subjected to Western blotting. Lane 1: control; lanes 2–4: VN/50-1 (1, 5 and 10 μ M); lanes 5–7: VN/14-1 (1, 5 and 10 μ M). Numbers below the blot show fold increase in expression of the protein as analysed by ImageQuant densitometry analysis. Membranes were stripped and probed for β-actin to verify equal protein loading. Primary antibody (Cell Signaling Technology) 1 : 1000 in 5% milk TBST overnight at 4°C and secondary antibody (anti-rabbit) 1 : 2000 for 1 h at RT. The experiments were repeated twice with similar results.