Figure 7
TbPth4 RNAi silencing in the dKO TbMrf1 background generates more severe phenotypes than the loss of TbMrf1 alone. (a,b) Growth curves of the noninduced (-tet) and induced (+tet) RNAi TbPth4 in either SM BF 427 (a) or dKO TbMrf1 cells (b) were measured for 14 days. Right panels: RT-qPCR analysis of the TbPth4 transcript levels after 3 (d3) or 5 (d5) days of tetracycline induction compared to noninduced RNAi cells. The relative changes in transcript abundance are plotted on a linear scale. β-tubulin transcript levels were used as internal controls. (c) The sedimentation profile of 12S and 9S rRNAs in dKO TbMrf1 + RNAi TbPth4 cells that were either never induced (d0) or induced with tetracycline for 3 (d3) or 5 (d5) days. BF427–8 μg of total RNA from BF 427. Input – 3 μg of total RNA isolated from the remaining material (if available) that was loaded on a gradient. (d) After normalization to a BF 427 RNA, the relative intensities of 9S and 12S rRNA signals from each sample were plotted. (e) The native F1- and FoF1-ATPase complexes were visualized using light blue native electrophoresis. dKO TbMrf1 7wk and dKO TbMrf1 + RNAi TbPth4 cells were either never induced (d0) or induced with tetracycline for 3 (d3) or 5 (d5) days. The F1-ATPase (F1) and the FoF1-ATPase monomer and dimer were all visualized using specific polyclonal antibodies against either F1-ATPase subunit β or Fo-ATPase subunit OSCP. (f) SDS-PAGE Western blot analyses of the same mitochondrial lysates as in (e). The steady state abundance of mt hsp70, F1-ATPase subunits β and p18 and Fo-ATPase subunits OSCP and ATPaseTb2 were determined using specific antibodies. (g) The ∆ψm of TMRE stained BF 427 and dKO TbMrf1 + RNAi TbPth4 cells that were either never induced (d0) or induced with tetracycline for 3 (d3) or 5 (d5) days was measured by flow cytometry. The median fluorescence for each sample is depicted on the y-axis of the column graph. The results are means ± s.d. (n = 5). *p < 0.02, Student’s t test.
