Figure 4

Modulation of apoptosis-related protein levels by GUT-70. MCL cells were treated with GUT-70 (JVM-2, 5 μ M; Granta 519, 5 μ M; Jeko-1, 1 μ M; MINO, 5 μ M) for the indicated times. (A) Cells were subjected to lysis, then apoptosis-related proteins and macroautophagy marker LC3 were analysed by western blot. Western blot images are representative results from three independent experiments. (B) Cells were harvested after 18 h treatment with GUT-70, and Noxa mRNA expression levels were detected by TaqMan RT–PCR analysis. The abundance of transcripts of Noxa relative to GAPDH transcripts was determined as described in Materials and Methods. Graphs show the representative data from two independent experiments with similar results. (C) Cells were treated with GUT-70 for 24 h, and Mcl-1 immunoprecipitation was performed as described in Materials and Methods. Total extracts were analysed by western blotting for Noxa. Western blot images are representative results from three independent experiments. (D) Cells were treated with GUT-70 for 24 h, then conformational changes in BAK were measured by intracellular flow cytometry as described in Materials and methods. To block the caspase activation-mediated conformational changes of BAK, cells were preincubated for 1 h with 100 μ M Z-VAD-FMK. Data represent duplicate experiments. ^*P<0.01; ^**P<0.05. RE, relative expression.