Figure 4.

Lack of functional ETC complexes in PTCD1 KO cells causes deficit in oxidative phosphorylation and is reversed by PTCD1 expression. A, Western blot detection of PTCD1 and subunits of the electron transport chain complexes in the lysates of WT and PTCD1 KO cells. The selected subunits are labile when unbound and are used to estimate the levels of assembled complexes. B, Quantification of ETC complexes in cell lysates as shown in A. C, PTCD1 expression in KO cells (PTCD1 KO#1) was restored by stably expressing PTCD1-Flag (with GFP-T2A-puromycin, RFP as control) and ETC complexes in two independent rescue cell lines (KO#1 +PTCD1 clone1, clone2) were assessed by Western blotting as in A. GFP detection was used to confirm equal transgene expression. Note that the Flag tag on PTCD1 slightly increases its MW. D–G, Summary of Seahorse assay experiments with WT and PTCD1 KO (KO#1 +RFP) cells and KO cells re-expressing PTCD1 (KO#1 +PTCD1 clone1, clone2). D, E Quantification of baseline, peak (ETC uncoupled), and spare (difference between basal and maximum) respiration. F, G, Glycolytic flux at baseline and upregulated in response to blocked mitochondrial ATP production (glycolytic reserve). Data for quantifications from four (B) or three (D–G) independent experiments and presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 between cell lines (B) by one-way ANOVA with post hoc Tukey's or versus KO#1 +RFP cells (D–G) by one-way ANOVA with post hoc Dunnett's multiple-comparison test. All OCR and ECAR values normalized to protein content.