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. Author manuscript; available in PMC: 2021 Jul 5.
Published in final edited form as: Sci Signal. 2021 Jan 5;14(664):eabc4436. doi: 10.1126/scisignal.abc4436

Figure 4. Activities and assembly of respiratory complexes were severely impaired or absent in FH−/− HLRCC tumor-derived cell lines.

Figure 4.

(A) In-gel NADH-dehydrogenase activity assay to assess Complex I (CI) activity was performed in five FH−/− HLRCC tumor-derived cell lines, two control cell lines (RPTEC and HRCE), a mtDNA-deficient clear cell RCC cell line UOK121rho0 and its respiration-competent parental cell line, UOK121. (B) Mitochondrial lysates from the cell lines indicated in (A) were analyzed by native IB for the nuclear-encoded NDUFS1 to assess Complex I levels and assembly. CI-CIII2-CIVn designates Complex I-containing supercomplexes which include dimeric Complex III and 1-4 copies of Complex IV. (C) Mitochondrial lysates from the cell lines indicated in (A) were analyzed by SDS IB for the nuclear-encoded CI subunit NDUFS1. (D) Mitochondrial lysates from the cell lines indicated in (A) were analyzed by native IB for the core Complex III subunit UQCRC1. CIII2-CIV and CIII2-CIVn are supercomplexes containing dimeric Complex III and 1-4 copies of Complex IV. (E) Mitochondrial lysates from the cell lines indicated in (A) were analyzed by SDS IB for the Complex III nuclear encoded subunit UQCRC1 and the mtDNA-encoded Complex III subunit MT-CYB. (F) In-gel cytochrome c oxidase assay to assess Complex IV activity was performed in the cell lines indicated in (A). (G) Mitochondrial lysates from the cell lines indicated in (A) were analyzed by native IB for the mtDNA-encoded Complex IV subunit MT-CO1 to assess Complex IV assembly. (H) Mitochondrial lysates from the cell lines indicated in (A) were analyzed by SDS IB for the mtDNA-encoded Complex IV subunits MT-CO1, MT-CO2 and MT-CO3. (I) Mitochondrial lysates from the cell lines indicated in (A) were analyzed by native IB (top) for the nuclear-encoded Complex V subunit ATP5A to assess Complex V assembly (F1/F0 ATPase complex). SDS IB for ATP5A and MT-ATP6 proteins (lower panels) were performed on the same samples. Data shown in (A) to (I) are representative of three or more independent experiments. (J) NDUFS1 mRNA expression levels in FH−/− tumor cell lines and controls. (K) MT-ND1 mRNA expression levels in FH−/− tumor cell lines and controls. Data in (J) to (K) were obtained from three independent biological replicates per group and expression levels normalized to RPTEC cells, mean % control ± SD. (L) qPCR measurement of mtDNA copy number in FH−/− tumor cell lines and controls, n=3-5 biological replicates per group, means ± SD.