Figure 2. The mitochondrial translation machinery regulates global mitochondrial activity and Eμ-myc cell growth.

A. Eμ-myc lymphoma cells were transduced with shRNAs against Ptcd3, Mrps5, Mrps27, Renilla luciferase (Ren) or a control empty vector (EV). The number of viable cells at each time point (top) was determined by trypan blue staining. For each single shRNA the efficiency of knockdown was evaluated by real-time qPCR three days post-transduction (bottom). Transcript abundance is expressed as mean ± s.d. of triplicate measurements, expressed relative to the EV control and normalized to Rplp0. B. Doubling time was calculated from the growth curves in A., using the formula described in experimental procedures. C. Cell death was evaluated by Annexin V and propidium iodide (PI) staining at the 48hrs time-point. The graph shows the percentage of Annexin V+/PI- (black) and Annexin V+/PI+ cells (grey) corresponding to early and late apoptotic cells, respectively. D. Western blot analysis of components of the Electron Transport Chain (ETC) Complexes I-IV following 48hrs of knock-down. As loading controls, we used antibodies against cytoplasmic Vinculin and the mitochondrial Voltage-dependent anion channel (VDAC). E. Real-time qPCR quantification of mtDNA, normalized to nuclear DNA. Results are shown as mean ± s.d. from triplicate measurements. F. Oxygen consumption rate (OCR) was determined as described in experimental procedures at 48hrs. Data were normalized to total cellular protein contents. G. Mitochondrial membrane potential was measured by staining with the cationic cyanine dye DilC1(5) for one representative shRNA per each mitochondrial ribosomal protein (MRP) at 48hrs. Values are expressed relative to the shRen control. All plotted values are the mean ± s.d. from three independent experiments. * p < 0.05; ** p < 0.01; ns, not significant, as determined by Student's t test. See also Supplementary Figures S1 and S2.