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. 2015 Oct 2;6(34):35667–35683. doi: 10.18632/oncotarget.5523

Figure 5. ABT-737 regulates Bim expression through post-translational modifications via the ERK1/2 signaling pathway.

Figure 5

MC-3 and HN22 cells were treated with or without ABT-737, and the mRNA levels of Bim were analyzed by RT-PCR A. HN22 cells were preincubated with a protein synthesis inhibitor CHX (0.01 μg/ml) for 1 hr prior to treatment with ABT-737 for 24 hr; afterwards, cells were harvested for Western blot analysis to detect Bim expression B. The expression levels of p-ERK1/2, total ERK1/2, and p-Bim (Ser69) were analyzed by Western blot C. HN22 cells were preincubated with a TPA (a phorbol ester, 20 ng/ml) for 30 min prior to treatment with ABT-737 for 12 h; afterwards, cells were harvested for Western blot analysis to detect p-ERK1/2, total ERK1/2, and p-Bim (Ser69) D. HN22 cells were co-incubated with a TPA (20 ng/ml) and ABT-737 for 12 hr;, after which nuclear fragmentation and condensation were evaluated by DAPI staining with fluorescence microscopy (magnification X400) E. Qualitative estimation of ABT-737-induced apoptotic cell death was obtained by a live/dead assay, which differentially labels live (green) and dead (red) cells with fluorescent dyes F. β-actin was used as an internal control. The results are shown as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.