Figure 5

BOT-4-one induces apoptosis by down-regulation of anti-apoptotic gene expression. (A) L540 cells treated with either vehicle (DMSO) alone or BOT-4-one (30 µM) for 48 h were harvested, stained with fluorescein isothiocyanate-conjugated BrdU antibody and propidium iodide (PI). The cells were subsequently subjected to flow cytometry. A representative flow cytometric analysis was shown and the percentages of TUNEL-positive cells were indicated. (B and E) L540 cells were treated with either vehicle (DMSO) alone or BOT-4-one for 48 h, whole cell extracts were prepared and immunoblot analysis was performed with antibodies specific for the molecules indicated. (C and D) Total RNA was extracted from L540 cells treated with BOT-4-one for 8 h, and real-time PCR was performed. Anti-apoptotic gene expression was represented with relative fold (C) or stained with EtBr after electrophoresis (D). Treatment with BOT-4-one increased cleaved fragments of PARP and caspase-3, an apoptotic hallmarks (arrows in B), and decreased the expression of anti-apoptotic genes such as Bcl-2, Bcl-xL, Mcl-1, and survivin (C-E). GAPDH served as a loading control.