FIGURE 8.

Analysis of mitochondrial function in SPS1/SelD knockdown cells. A, analysis of mitochondrial gene expression. mRNA levels of cytochrome b, ND5, and COI, were examined by northern blotting. The film was exposed for 48 h. Probes are shown on the left of the autoradiogram. Treatment times and types of dsRNAs used in treating cells are shown at the top. Although SPS1/SelD mRNA was ablated, the level of mitochondrial RNAs was not changed by SPS1/SelD knockdown. B, Southern hybridization analysis of mitochondrial and nuclear DNA. Total genomic DNA containing mitochondrial DNA was prepared and digested with restriction enzyme, and Southern hybridization was performed using ND5 (mitochondrial gene) and rp49 (nuclear gene) genes as probes. The film was exposed for 48 h. Neither the degradation of mitochondrial or nuclear DNA nor reduction of DNA content was observed in SPS1/SelD knockdown cells. C, measurement of intracellular ATP levels. Cellular ATP levels were measured in dsRNA-treated cells as described under “Experimental Procedures.” The level of ATP was normalized to the protein contents. Oligomycin, an inhibitor of ATP synthase, was added to the cells to inhibit the function of mitochondria and used as a negative control for ATP synthesis. Experiments were performed in triplicate. Standard deviations from the mean are indicated by error bars.