Fig. 6

IFI6 modulates mitochondrial ATP production and the oxidative phosphorylation efficiency. a. ESCC patients in the TCGA database were divided into a high-IFI6 group and a low-IFI6 group according to their IFI6 expression level. GSEA was performed to compare the two groups. NES: normalized enrichment score. b. Quantitative results of ATP production in ESCC cells. Total ATP levels were measured in the indicated cells via a luminescence assay. Data were normalized to the ATP level in ShControl cells and are presented as the means and SDs (n = 3). Statistical significance was determined by a two-tailed Student’s t-test. **P < 0.01. c. Representative plots (upper) and quantitative results (bottom) of the cellular OCR, basal and maximal respiration rates in the different groups. The indicated ESCC cells were subjected to extracellular flux analysis in the Seahorse XF instrument. The arrows and dotted lines indicate the addition of Oligo (oligomycin) (1 μM), FCCP (Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) (0.5 μM) and Rot&AMA (Rotenone and Antimycin A) (0.5 μM each). The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. **P < 0.01. d. Representative plots (upper) and quantitative results (bottom) of the real-time ECAR, glycolysis and glycolytic capacity assays in the indicated ESCC cells. The ECAR was determined following sequential addition of glucose (10 mM), oligomycin (1 μM) and 2-DG (100 mM). Glycolysis was measured by subtracting the ECAR after glucose addition from the ECAR before glucose addition. The glycolytic capacity was calculated by subtracting the ECAR after oligomycin treatment from the ECAR before glucose addition. The data are presented as the means and SDs (n = 3). Statistical significance was determined by a two-tailed Student’s t-test. e. Representative plots (left) and quantitative results (right) of the complex I-dependent OCR in the different groups. Pyruvate (Pyr) (5 mM) and malate (Mat) (5 mM) were added to digitonin (Dig)-permeabilized cells, and the OCR was monitored. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. **P < 0.01. f. Representative plots (left) and quantitative results (right) of the complex III-dependent OCR in the different groups. Rotenone was added to digitonin-permeabilized cells to inhibit complex I, after which G3P (5 mM) was added, and the OCR was monitored. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. **P < 0.01. g. Representative plots (left) and quantitative results (right) of the complex II-, and complex IV-dependent OCRs in the different groups. Rotenone (1 μM) was added to inhibit complex I; succinate (Suc) (5 mM), Antimycin (AMA) (1 μM) and TMPD/ascorbate (500 μM and 5 mM, respectively) were then added to digitonin-permeabilized cells, and the OCR was monitored. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. **P < 0.01