Figure 4.

Representative changes in the respiratory activity and the transcripts and protein levels of mitochondrial respiratory complexes. (A) Mitochondrial complexes activity was analyzed by Seahorse XF24 analyzer. (B) The differential changes in the transcripts of mitochondria-encoded respiratory complex genes (ND1, CYTB, COI and ATP6) and nucleus-encoded respiratory complex genes (NDUFB8, SDHB, UQCRC2 and ATP5A) were measured by qPCR. The expression levels were normalized to an internal control (ACTB) and fold changes were relative to the control. (C) Immunoblots of mitochondrial respiratory complex proteins are shown. Antibody cocktails (MS604) against mitochondrial complex subunits were used to examine the expression profiles of mitochondrial respiratory complexes. The five distinct bands in the immunoblots represent Complex I subunit NDUFB8 (~20 kD, nuclear encoded), Complex II subunit (~30 kD, nuclear encoded), Complex III subunit Core 2 (~47 kD, nuclear encoded), Complex IV subunit I (~39 kD, mitochondrial encoded), and ATP synthase subunit alpha (~53 kD, nuclear encoded). All the proteins were increased in the mitochondrial-encoded or nuclear-encoded mitochondrial respiratory complex proteins. β-Actin was used as a loading control. (D) Fold change in mitochondrial DNA (mtDNA) copy number relative to that of PE089 cells. ND1 was used to represent the mtDNA copy number. B2M was used as an internal control gene of nuclear DNA. The values represent the mean ± S.D. from three independent experiments and each experiment performed in duplicate. * p < 0.05. ** p < 0.01. *** p < 0.001.