Figure 1

Depletion of Drosophila ClpP protease in Schneider S2 cells. Schneider S2 cells with no plasmid (control) or carrying pMt/invGFP/Hy (GFP RNAi) or pMt/invClpP/Hy (ClpP RNAi) were cultured for 5 d in the presence or absence of 0.1 mM CuSO4. (A) Immunoblot analysis was carried out as described in Materials and Methods. (B) Relative protein ratios of ClpP and mtTFB2 was quantitated as described in Materials and Methods. The results represent the mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001 versus control cells without induction (Student’s t-test). (C) Northern blot analysis of the levels of mitochondrial genome encoded transcripts in ClpP depleted Schneider S2 cells. The nuclear genome encoded transcripts, RP49 and TubA, was used as a loading control. The results are representative of two independent experiments. (D) The relative ratio of mtTFB2 mRNA was quantitated as described in Materials and Methods. The results represent the mean ± SD of three independent experiments and **P < 0.01; ***P < 0.001 versus control cells without induction. (E) The relative mtDNA copy number was quantitated as described in Materials and Methods. The results represent the mean ± SD of three independent experiments and *P < 0.05 versus control cells without induction (Student’s t-test). (F) Mitochondrial translation products were labeled in Schneider S2 cells after 5 d of culture in the presence or absence of 0.1 mM CuSO₄. Labeling was carried out with EXPRE³⁵S³⁵S protein labeling MIX for 45 min in the presence of emetine and cycloheximide, and then total cell lysates were prepared. Coomassie Brilliant Blue (CBB) staining was used to check for equal loading of the samples. Left panel, aliquots containing 10 μg of total protein each were fractionated by 15–20% gradient SDS-PAGE. Right panel, the relative mitochondrial protein synthesis ratio of ClpP-depleted cells. Schneider S2 cell lines were cultured for 5 d in the presence or absence of 0.1 mM CuSO4. The relative mitochondrial protein synthesis ratio was quantitated by average of the radiolabeled products of mtDNA encoded subunits, COX1 and COX2. The results represent the mean ± SD of three independent experiments and *P < 0.05; **P < 0.01 versus control cells without induction (Student’s t-test). Full blots are presented in Supplementary Fig. S9.