Figure 1. Mitochondrial alterations ensuing the downregulation or overexpression of the c subunit of the FO ATP synthase in HeLa cells. (A and B) Human cervical carcinoma HeLa cells were either transfected with a control siRNA (siSCR) or a mix of siRNAs targeting ATP5G1, ATP5G2 and ATP5G3 (siATP5G) for 48 h (A) or, alternatively, subjected to mock transfection or transfected with a plasmid encoding MYC-tagged ATP5G1 for 24 h (A and B), and then processed for either the immunoblotting-assisted detection of the FO c subunit, the FO a subunit, the F1 α subunit, cytochrome c (Cyt. c) and voltage-dependent anion channel 1 (VDAC1) (A) or the immunofluorescence-microscopy assisted visualization of heat shock 60 kDa protein 1 (HSPD1) and the FO c subunit (via the MYC tag). In (A) (reporting representative results), β actin levels were monitored to ensure the equal loading of lanes. (C and D) HeLa cells were transfected as in (A and B) but in combination with a plasmid coding for a mitochondrially targeted variant of luciferase, then stimulated with 100 μM histamine (Hist) and monitored for light emission over time upon the exogenous administration of luciferin. Representative traces as well as quantitative data illustrating the Hist-induced increase in luminescence (means ± SEM, n = 6) are reported. *p < 0.05 (unpaired Student’s t-test), as compared with equally stimulated, mock-transfected cells; n.s. = non-significant (unpaired Student’s t-test), as compared with equally stimulated, siSCR-transfected cells. (E) HeLa cells transfected as in (A and B) and then maintained in baseline conditions were stained with tetramethylrhodamine methyl ester (TMRM) for the assessment of mitochondrial transmembrane potential (ΔΨm). Quantitative data (means ± SEM, n = 5) are reported. *p < 0.05 (unpaired Student’s t-test), as compared with mock-transfected cells; n.s. = non-significant (unpaired Student’s t-test), as compared with siSCR-transfected cells. (F) HeLa cells were transfected as in (A and B) but in combination with a plasmid encoding a mitochondrially targeted variant of GFP, then maintained in control conditions and analyzed by 3D deconvolution fluorescence microscopy. Representative images and quantitative data illustrating the number of GFP+ 3D objects per cell (means ± SEM, n = 7) are reported. *p < 0.05 (unpaired Student’s t-test), as compared with mock-transfected cells; n.s. = non-significant (unpaired Student’s t-test), as compared with siSCR-transfected cells.