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. 2020 Aug 31;130(10):5245–5256. doi: 10.1172/JCI135479

Figure 2. HSCB depletion decreases Fe-S protein expression and impairs respiration.

Figure 2

(A–D) Western blots of Fe-S cluster biogenesis, Fe-S, and lipoylated proteins (A) and respiratory proteins (B) in K562 erythroleukemia cells 10 days after control and HSCB-specific shRNA infection. Three independent experiments are shown. Western blots in A and B are quantified in C and D, respectively. Scrambled control (shSC) and HSCB‑specific shRNAs 1 (sh1) and 2 (sh2) are indicated by black, red, and blue, respectively. NI, noninfected. For all panels, mean expression ± SD is normalized to NI cells. ANOVA, *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. shSC. (E–G) Seahorse Extracellular Flux (XF) Analyzer Cell Energy Phenotype analyses of shRNA-treated K562 cells. Data presented are representative of at least 3 independent experiments. NI and shSC-treated K562 cells are indicated by black points with long-dashed and short-dashed lines, respectively. sh1- and sh2-treated K562 cells are indicated by red and blue, respectively. (E) Cell Mito Stress Test energy maps of basal (left points) and stressed (right points) oxygen consumption versus extracellular acidification, which is a measure of glycolysis. (F) Seahorse extracellular flux analysis. Oligomycin (O), carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), and rotenone/antimycin A (R/A) allow assessment of basal ATP production, maximal respiration, and non-mitochondrial respiration, respectively. (G) Basal respiration and residual respiratory capacity. Activity normalized to NI cells. ANOVA, ****P < 0.001 vs. shSC. ACO2, aconitase 2; ATP5A, ATP synthase F1 subunit α; COXII, mitochondrially encoded cytochrome c oxidase II; CS, citrate synthase; FECH, ferrochelatase; HSPA9, heat shock protein family A (Hsp70) member 9; KGDH, α-ketoglutarate dehydrogenase; MTCYB, mitochondrially encoded cytochrome b; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8; PDH, pyruvate dehydrogenase; RC, respiratory complex; SHDA and SDHB, succinate dehydrogenase A and B subunits; UQCRC2, ubiquinol–cytochrome c reductase core protein 2. E2 subunits of PDH and KGDH were detected with an anti–lipoic acid antibody.