Research Paper Volume 13, Issue 5 pp 7300—7313

Maf1 suppression of ATF5-dependent mitochondrial unfolded protein response contributes to rapamycin-induced radio-sensitivity in lung cancer cell line A549

Rapamycin suppresses Akt-mediated Maf1 re-phosphorylation in response to IR in A549 cells. (A, B) Maf1 phosphorylation (P) was decreased by IR but was re-phosphorylated after 8 hours (Post IR). Rapamycin inhibited Maf1 re-phosphorylation. A549 cells were irradiated (IR) at 6 Gy then treated with 100 nM rapamycin (Rap) for 48 hours. Maf1 phosphorylation was detected by slow migration by Western blot. (C, D) Akt phosphorylation at Ser473 was increased by IR. Akt phosphorylation was detected with phospho-specific antibody. (E, F) Akt was required for Maf1 re-phosphorylation in response to IR. A549 cells with stable Akt knockdown were analyzed by Western blot at indicated time points. In all panels, representative data and the quantifications of at least 2 biological repeats are shown. Data were normalized to time 0. The error bars stand for Standard Deviation (SD) of the mean. Statistical significance was evaluated by 2-tailed, paired student’s t-test (ns, not significant, *, P

Figure 2. Rapamycin suppresses Akt-mediated Maf1 re-phosphorylation in response to IR in A549 cells. (A, B) Maf1 phosphorylation (P) was decreased by IR but was re-phosphorylated after 8 hours (Post IR). Rapamycin inhibited Maf1 re-phosphorylation. A549 cells were irradiated (IR) at 6 Gy then treated with 100 nM rapamycin (Rap) for 48 hours. Maf1 phosphorylation was detected by slow migration by Western blot. (C, D) Akt phosphorylation at Ser473 was increased by IR. Akt phosphorylation was detected with phospho-specific antibody. (E, F) Akt was required for Maf1 re-phosphorylation in response to IR. A549 cells with stable Akt knockdown were analyzed by Western blot at indicated time points. In all panels, representative data and the quantifications of at least 2 biological repeats are shown. Data were normalized to time 0. The error bars stand for Standard Deviation (SD) of the mean. Statistical significance was evaluated by 2-tailed, paired student’s t-test (ns, not significant, *, P<0.05, **, P<0.01).