Figure 5

Aloperine-mediated inhibition of clonogenic survival is irreversible. (A) Cells were treated with aloperine (ALO) for 9 days to inhibit clonogenic survival. Cells were then washed and cultured in a drug-free medium for an additional 9 days (ALO-9 days-washed) or continuously cultured in a drug-containing medium for an additional 9 days (ALO-18 days), followed by colony staining. (B) Cells were treated by aloperine for 48 h, washed at the indicated times and harvested for Western blotting analysis. (C) Aloperine displays a synergistic effect with TRAIL. U266 (left on panel) and MM.1S (right on panel) cells were treated for 48 h with (red bars) or without (blue bars) aloperine, followed by the addition of the indicated concentrations of TRAIL for 48 h. Apoptosis was determined via FACS analysis of DNA fragmentation of propidium iodide-stained nuclei. Data represent the mean ± SD of at least three independent experiments carried out at least in duplicate; ^a P < 0.001. GAPDH was assessed by Western blotting. All protein levels were quantified densitometrically and normalized to GAPDH. (D) U266 and MM.1S cells were treated with 80 μM aloperine and aloperine (80 μM) + borterzomib (2.5 nM) for 48 h, harvested, and analyzed by flow cytometry.