Figure 5
From: PGC-1α buffers ROS-mediated removal of mitochondria during myogenesis

FOXO1 promotes mitophagy in a ROS-dependent manner during myogenesis. C2C12 cells were transfected with scramble (scr) or PGC-1α siRNA (PGC-1α(−)) and differentiated in DM for the indicated days. (a) Twenty micrograms of total proteins were loaded for western blot analysis of FOXO1. TUBB was used as the loading control. (b) Twenty micrograms of nuclear and cytoplasmatic proteins were loaded for western blot analysis of FOXO1. Sp1 and LDH were used as markers of fraction purity and as the loading control. (c, d) At day 2 of myogenesis, ChIP assay was carried out on cross-linked nuclei using LC3II (upper panel) or PINK1 (bottom panel) antibody followed by qPCR analysis of FOXO1 consensus sequence. Data are expressed as means±S.D. (n=4, *P<0.001 versus scr cells; °P<0.001 versus day 0 PGC-1α(−) cells). (e) C2C12 cells were treated with 200 μM Trolox and maintained throughout the experiment (2 days). Twenty micrograms of nuclear and cytoplasmatic proteins were loaded for western blot analysis of FOXO1. Sp1 and LDH were used as markers of fraction purity and as the loading control. (f, g) C2C12 cells were treated with 200 μM Trolox and maintained throughout the experiment (2 days). ChIP assay was carried out on cross-linked nuclei using LC3II (upper panel) or PINK1 (bottom panel) antibody followed by qPCR analysis of FOXO1 consensus sequence. Data are expressed as means±S.D. (n=6, *P<0.001 versus scr cells; °P<0.001 versus untreated day 2 PGC-1α(−) cells). Immunoblots reported in the figures are representative of at least four experiments that gave similar results