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. 2018 Oct 4;8:388. doi: 10.3389/fonc.2018.00388

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Copyright © 2018 Briston, Stephen, Thomas, Esposito, Chung, Syafruddin, Turmaine, Maddalena, Greef, Szabadkai, Maxwell, Vanharanta and Ashcroft.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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pVHL re-expression regulates mitochondrial protein expression. (A) Western blot analysis of mitochondrial protein expression in 786O-VHL (VHL, stably re-expressing pVHL) cells and 786O-EV (EV, vector control) cells. α-tubulin and β-actin were used as load controls. Densitometric analysis of western blots show fold change in mtCO-2 and COXIV protein levels normalized to β-actin and α-tubulin load controls respectively. (B) Graph shows relative expression of mRNA transcripts (ND6, SDHA, cytochrome b, mtCO-2, and COX IV) in 7860-VHL (VHL) and 786O-EV (EV) cells, measured using RT-qPCR. Data were analyzed using the comparative Ct method (n = 4). (C) Boxplot of CHCHD4 expression in VHL mutated and VHL non-mutated ccRCC. TGCA-KIRC data was downloaded from cBioportal. n = 534 patients were divided into VHL mutant (n = 202) and VHL non-mutant (n = 332) and analyzed for CHCHD4 expression in each group. Mean expression VHL non-mutated = 235.8, VHL mutated = 212.0. Median values are shown for each group (black line), vertical size of the boxes are the interquartile range (IQR), and whiskers represent 1.5 × IQR. Data were analyzed using a t-test with Welch's correction p = 0.00034. (D) Western blots show CHCHD4 protein expression in total (Load), cytoplasmic (Cyto), and mitochondrial (Mito) fractions from 786O-VHL (VHL) and 7860-EV (EV) cells. HCT116 cells transiently expressing non-silencing control siRNA (NSC) or CHCHD4 siRNAs (CH), were used as controls for CHCHD4 protein expression (left two lanes). α-tubulin and VDAC1 were used as cytoplasmic and mitochondrial protein markers respectively. (E) Graph shows relative expression of CHCHD4 mRNA expression in 7860-VHL and 786O-EV cells, measured using RT-qPCR. Data analyzed using the comparative Ct method. Data are presented as mean ± S.E.M. n = 4 (not significant (n.s.) p > 0.05). (F) Western blots show expression of mitochondrial proteins CHCHD4 and respiratory chain subunits NDUFB10 (CI), SDHA (CII), UQCRC2 (CIII), and COX IV (CIV) in 786O-EV cells transiently expressing non-silencing control siRNA (NSC, 20 nM) or two independent CHCHD4 siRNAs (CH1 and CH2, 20 nM). α-tubulin and β-actin were used as a load controls. (G) Relative expression of CHCHD4 mRNA in cells described in (A), measured using RT-qPCR. Data were analyzed using the comparative Ct method. Data are presented as mean ± S.D. n = 3 (**p < 0.01).