Fig. 4: PZL-A stimulates mtDNA synthesis and OXPHOS activity in patient cells.
From: Small molecules restore mutant mitochondrial DNA polymerase activity
a, Schematic illustration of the mtDNA depletion and recovery experiment. Drawing by Jennifer Uhler (copyright holder). b–d, mtDNA depletion and repopulation in wild-type (b), POLGA467T/G848S (c) and POLGW748S/R232H fibroblasts treated with 50 ng ml−1 EtBr for 4 days. PZL-A and vehicle were added after EtBr removal. mtDNA was quantified at the indicated time points (mean ± s.d.; n = 3 biological replicates). e, Dose-dependent recovery of mtDNA in POLGA467T/G848S mutant cells after 7 days of EtBr treatments (50 ng ml−1), followed by recovery for 10 days. PZL-A was added at the indicated concentrations after removal of EtBr. Southern blot analysis after BamHI digestion was used to detect mtDNA and 7S DNA. Nuclear 18S rDNA was used as a loading control. f, As in e, but with POLGW748S/R232H mutant cells, 4 days of EtBr treatments and recovery for 5 days. g, PZL-A (1 μM) stimulates mtDNA synthesis in intact mitochondria isolated from patient-derived fibroblasts. DNA synthesis was monitored by incorporation of radiolabelled dCTP. VDAC was used as a loading control. h, Immunoblot analysis of OXPHOS complex subunits in mutant fibroblasts after 7 days recovery. PZL-A (1 μM) increased NDUFB8 (complex I (CI)), UQCRC2 (complex III (CIII)) and COXI (complex IV (CIV)) levels, whereas SDHB (complex II (CII)) and ATP5A (complex V (CV)) remained unchanged. Tubulin was used as a loading control. i–k, Mitochondrial respiration in fibroblast cells treated as in a. A Seahorse XFe96 pro extracellular flux analyser was used to measure basal respiration (i), maximal respiration (j) and ATP production (k) after 7 days of recovery. Data are mean ± s.d. (n = 3 biological replicates). Unpaired two-tailed Welch’s t-tests were used to determine P values. Blots in e–h are representative of n = 2 independent experiments. For source data of gels and blots in e–h, see Supplementary Fig. 8.
