Fig. 3. mtDNA, mitochondrial transcript, and OXPHOS protein levels in POLRMT patient fibroblasts.

a Effects of the patient variants on mtDNA levels in vivo were analysed by Southern blot (left panel) and qPCR (right panel). No significant deletions/depletion of mtDNA were observed in P1, P2, and P6 while mild mtDNA depletion was observed in P3 when compared to control (Ct). Southern blotting was repeated three times and a representative example is shown. For qPCR analysis, 6 technical repeats were performed (patient cells were grown in two separate cell cultures, which were each analysed three times). b Levels of mitochondrial transcripts in patient fibroblasts were analysed by RT-qPCR. P1, P2, and P6 showed a mild down-regulation of gene expression whereas P3 showed a severe loss of mitochondrial transcripts (5–32% of wild-type levels). For qPCR analysis, 4–6 technical repeats were performed (patient cells were grown in two separate cell cultures, which were each analysed 2–3 times). c Western blot analysis of whole-cell lysates extracted from adult control (Ct1, Ct2) and POLRMT patient (P1, P2, P3, P6) fibroblasts (left panel). P1 and P3 showed a decrease in the steady-state levels of subunits of CI (NDUFB8), CIII (UQCRC2), and CIV (COXII). Normal levels of OXPHOS subunits were detected in P2 and P6. One-dimensional BN PAGE analysis revealed a combined OXPHOS assembly defect in P3 and a slight defect in the assembly of CIV in P1 (right panel). The nuclear-encoded SDHA and/or SDHB subunits were used as loading controls. Representative blots of three independent SDS and BN PAGE experiments for P1, P2, and P3 are shown. One representative western blot experiment out of two independent analysis is shown for P6.