Figure 3
Respiratory complex II, but not complexes III and IV, is upregulated in XP-C cells. (A) OCR was measured using 4 × 106 cells in standard reaction medium. Cells were permeabilized with 30 µM digitonin, with the following sequential additions: 5 mM succinate, 0.25 µM CCCP, 1 µM AA and 200 µM TMPD + 2 mM ascorbate. The figure shows a representative trace OCR experiment. (B) The rates of oxygen consumption were calculated from data obtained as shown in panel A. (C) CII + CIII activity was measured using 12 µg of crude mitochondrial protein. Reduction of cyt c was monitored at 550 nm at 37 °C for 5 min. It was only considered the range where slopes of the reactions were linear. (D) Expression analysis of SDHA and SDHB genes by RT-qPCR. Fold-change in mRNA expression of unXP-C relative to the WT cell line. ACTB was set as reference gene for all genes analyzed. (E) Western blotting of CI subunit SDHB and ATPase subunit F1α. SDHB protein expression level was normalized against F1α in unXP-C relative to corrXP-C cells. The data represent mean ± SD of 3 independent experiments. *p < 0.05 and **p < 0.01.
