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. 2015 Nov;21(11):1873–1884. doi: 10.1261/rna.052365.115

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© 2015 Popow et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 4. — Metabolic analysis of cells depleted of FASTKD2. (A) Oxygen consumption rate (OCR) of HEK293 cells depleted of FASTKD2 by RNAi ([siCtrl] control siRNA; [siFASTKD2] FASTKD2 targeting siRNA pool; [Basal] basal respiration; [Oligo] leak respiration in the presence of oligomycin; [FCCP1] uncoupled respiration in the presence of 200 nM FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone); [FCCP2] uncoupled respiration in the presence of 250 nM FCCP; [A + R] residual respiration in the presence of antimycin a and rotenone). (B) Extracellular acidification rate (ECAR) of cells depleted of FASTKD2 by RNAi. Maximum ECAR measured in the presence of oligomycin (Oligo) was set to 100%. (C) Confirmation of RNAi-mediated depletion of FASTKD2 by Western blot. (D) Metabolic labeling of de novo synthesis of mitochondrial proteins in cells depleted of FASTKD2 by RNAi (left panel) or CRISPR-mediated mutagenesis (right panel) in the presence of emetine. (CBB) Coomassie brilliant blue stain. (E) OCR of HEK293 cells depleted of FASTKD2 by CRISPR. Abbreviations are as in A. (F) ECAR of cells depleted of FASTKD2 by CRISPR. (G) Relative growth of cells depleted of FASTKD2 by CRISPR in galactose media. (H) Western blot analysis of expression of FASTKD2 in crude mitochondrial preparations isolated from cells depleted of FASTKD2 by CRISPR. (I) Activity of ETC complexes in cells depleted of FASTKD2 by CRISPR. Values were normalized by the activity of citrate synthase and are stated for both cells depleted of FASTKD2 by CRISPR-mediated mutagenesis and Cas9-expressing control cells. (J) mtDNA levels of cells depleted of FASTKD2 by CRISPR. Values are stated as relative ratios of COXI (mitochondrial) to β-actin (nuclear) amplicons as determined by quantitative reverse transcriptase PCR.