Figure 4
From: PGC-1α buffers ROS-mediated removal of mitochondria during myogenesis

PGC-1α downregulation is associated with an increase of oxidative stress and alterations of mitochondrial homeostasis. 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 derivatized with DNP and carbonylation was detected by western blot with DNP antibody. TUBB was used as the loading control. (b) Twenty micrograms of total proteins were loaded for western blot analysis of SOD2 and TRX1. TUBB was used as the loading control. (c) C2C12 cells were treated with 200 μM Trolox and maintained throughout the experiment. ROS production was assayed by cytofluorimetric analysis after DCF-DA staining. ROS level was reported as the percentage of DCF-positive cells and expressed as means ±S.D. (n=3; *P<0.001 versus scr cells; °P<0.01 versus PGC-1α(−) cells). (d) Twenty micrograms of total proteins were derivatized with DNP and carbonylation was detected by western blot with DNP antibody. TUBB was used as the loading control. (e) C2C12 cells were incubated with MitoTrackerRed for 30 min and mitochondrial content was assayed by cytofluorimetric analysis. Data are expressed as percentage of MitoTrackerRed-positive cells (n=4; *P<0.001 versus scr cells; °P<0.01 versus PGC-1α(−) cells). (f) DNA was extracted and relative mtDNA content was assayed by analyzing the D-Loop level through qPCR. D-Loop value was normalized to RPL. Data are expressed as means±S.D. (n=5, *P<0.001 versus scr cells; °P<0.001 versus PGC-1α(−) cells). (g) Twenty micrograms of total proteins were loaded for western blot analysis of LC3I-II and PINK1. TUBB was used as the loading control. Immunoblots reported in the figures are representative of at least four experiments that gave similar results. (h) C2C12 cells were transfected with empty vector (Mock) or pSV-PGC-1α vector (PGC-1α(+)) and differentiated in DM for the indicated days. Twenty micrograms of total proteins were loaded for western blot analysis of PGC-1α, PARK2, PINK1 and BNIP3. TUBB was used as the loading control