Complementation and RNAi Studies
(A) Immunoblot analysis of one-dimension BNGE in immortalized fibroblasts from two controls (Ct1, Ct2) and mutant subjects S1 and S2. Samples were prepared using 10% w/v dodecylmaltoside. We used an antibody against NDUFA9 to detect complex I (I), an antibody against the subunit α of ATP synthase to detect complex V (V), an antibody against SDHA for complex II (II), an antibody against core I for complex III (III), and an antibody against subunit COX4 for complex IV (IV). Note that the amounts of cIV holocomplex and cIV+cIII2 supercomplex are clearly decreased in mutant samples compared to controls.
(B) Immunoblot analysis of one-dimension BNGE in immortalized fibroblasts from a control (Ct), mutant S2, and S2 stably transduced with APOPT1-HA. Note that the amounts of cIV and cIV+cIII2, which are markedly decreased in S2, are significantly increased in S2+APOPT1-HA.
(C) Quantitative densitometric analysis of the results obtained by three independent BNGE experiments. The intensities of complex IV (CIV) and supercomplex III + IV (CIII+CIV) signals were normalized to complex II; the ratio obtained in control fibroblasts was set as 100%. The p values were obtained by unpaired, two-tail Student’s t test. Bars represent standard deviations.
(D) Quantitative PCR analysis of APOPT1 transcript in naive, nontransduced immortalized fibroblasts, fibroblasts transduced with the “empty” vector pLKO.1, and with APOPT1-specific shRNA-2 and shRNA-3. The amount of APOPT1 transcript is decreased to approximately <5% by both shRNA, compared to the amount found in naive, nontransduced cells.
(E) Growth curves in immortalized fibroblasts from a naive control cell line, and the same cell line transduced with empty vector (pLKO.1), shRNA-2, and shRNA-3. Cell lines with severe APOPT1 knockdown (down to around 1% of the control mRNA levels 48 hr postinfection) show significantly decreased cell growth.