FIGURE 3.

Effect of CCCP treatment on mitochondrial respiratory chain expression and function. A, U2OS cells were allowed to grow for 24 h and then either untreated (−) or treated (+) with CCCP for 16 h. The effect of CCCP treatment on the steady-state levels of key transcription factors associated with the biogenesis of mitochondria and the expression of the respiratory chain was measured by immunoblotting. B, wild-type U2OS cells, along with the control shRNA and PRC shRNA1 lentiviral transductants, were plated and treated with CCCP as in A. The indicated subunits from each of the five respiratory complexes were detected by immunoblotting using a mixture of mouse monoclonal antibodies directed against each subunit. Subunit designations for the respective complexes (I–V) are indicated at the left, and gene names along with their nuclear (n) or mitochondrial (m) assignment are indicated at the right. The middle panel is a lower exposure of the upper panel, and the bottom panel shows PRC expression. C, the activity of respiratory complex I was measured in cell extracts using a dipstick assay (MitoSciences) as described under “Experimental Procedures.” Membranes were scanned for densitometric analysis, and the relative enzyme activity normalized to the untreated control is expressed as the averages ± S.E. for at least three separate determinations. D, lactate in the culture medium was measured enzymatically using an NADH-coupled reaction.