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

Maf1 is required for rapamycin to increase radio-sensitivity in A549 cells. (A–C) Maf1 mRNA (A) and protein levels (C) were knocked down by siRNA and shRNA. Representative western blot results are shown in (B). Experiments were performed for ≥3 times with replicates. Data were normalized to non-transfected control (Ctrl) and expressed as fold change. (D, E) Maf1 is required for rapamycin to increase apoptosis in A549 cells in response to radiation. Maf1 was knocked down by siRNA (si) in A549 cells. Cells were then irradiated (IR) with 6 Gy x-ray and treated with 100 nM rapamycin (Rap) as indicated. Apoptosis (Annexin V) and cell death (PI) were analyzed by flow cytometry after 48 hours. Experiments were performed for 2 times and representative results (D) and the quantifications of apoptotic cells (E) are shown. (F, G) Maf1 is required for rapamycin to enhance radiosensitivity in A549 cells. Cells treated with radiation and rapamycin were plated at 500 cells/plate and irradiated (+IR). Non-irradiated cells were plated at 50 cells/plate as controls (-IR). Colonies were counted after 2 weeks of incubation. Experiments were performed for 3 times and representative results are shown in (F) and the quantifications in (G). In all panels, 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 1. Maf1 is required for rapamycin to increase radio-sensitivity in A549 cells. (AC) Maf1 mRNA (A) and protein levels (C) were knocked down by siRNA and shRNA. Representative western blot results are shown in (B). Experiments were performed for ≥3 times with replicates. Data were normalized to non-transfected control (Ctrl) and expressed as fold change. (D, E) Maf1 is required for rapamycin to increase apoptosis in A549 cells in response to radiation. Maf1 was knocked down by siRNA (si) in A549 cells. Cells were then irradiated (IR) with 6 Gy x-ray and treated with 100 nM rapamycin (Rap) as indicated. Apoptosis (Annexin V) and cell death (PI) were analyzed by flow cytometry after 48 hours. Experiments were performed for 2 times and representative results (D) and the quantifications of apoptotic cells (E) are shown. (F, G) Maf1 is required for rapamycin to enhance radiosensitivity in A549 cells. Cells treated with radiation and rapamycin were plated at 500 cells/plate and irradiated (+IR). Non-irradiated cells were plated at 50 cells/plate as controls (-IR). Colonies were counted after 2 weeks of incubation. Experiments were performed for 3 times and representative results are shown in (F) and the quantifications in (G). In all panels, 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, ***, P<0.001, ****, P<0.0001).