Figure 1 | Cell Death & Disease

Figure 1

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

Figure 1

Nuclear and mitochondrial PGC-1α induction is necessary to mitochondrial functionality during myogenesis. C2C12 cells were differentiated in DM for the indicated days. (a) Twenty micrograms of total proteins were loaded for western blot analysis of PGC-1α, TFAM and COX4I1. TUBB was used as the loading control. (b) Total RNA was isolated, and relative mRNA level of PGC-1α was analyzed by RT-qPCR. Data are expressed as means±S.D. (n=6, *P<0.001). (c) Twenty micrograms of mitochondrial proteins extracts were loaded for western blot analysis of PGC-1α and TFAM. TOMM20 was used as the loading control. To exclude the presence of nuclear contaminants, the nitrocellulose was probed with H2B antibody. Numbers indicate the density of immunoreactive bands calculated using the Software Quantity one (Bio-Rad) and reported as the ratio of PGC-1α/TOMM20 and TFAM/TOMM20. (d) mtDNA-IP assay was carried out on cross-linked mitochondria using PGC-1α (left panel) or TFAM (right panel) antibody followed by qPCR analysis of D-Loop TFAM consensus sequence. Data are expressed as means±S.D. (n=4, *P<0.001). (e) C2C12 cells were transfected with scramble (scr) or PGC-1α siRNA (PGC-1α(−)), differentiated in DM for the indicated days. DNA was extracted and relative mtDNA content was assayed by analyzing D-Loop level through qPCR. D-Loop value was normalized to ribosomal protein large subunit (RPL). Data are expressed as means±S.D. (n=5, *P<0.001 versus day 0 scr cells; °P<0.001 versus scr cells). (f) Cells were grown on glass coverslips, differentiated until day 2 and incubated for 30 min with 1 μg/ml JC-1. Paraformaldehyde-fixed cells were stained with Hoechst 33342 and subjected to fluorescence microscopy. Magnitude insets are shown at the bottom and top of each picture. Images reported are from one experiment representative of three that gave similar results. Immunoblots reported in the figures are representative of at least four experiments that gave similar results

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