Fig 2: High glucose promotes cellular metabolism involving stimulation of c-Myc localization to mitochondria and its interactions with IF1. (A–F) OXPHOS (A–C, F) and glycolysis (D–F) activities were evaluated by Agilent Seahorse XFe24 Analyzer before and after additions of oligomycin (Oligo, 2 µM), FCCP (0.2 µM), rotenone plus antimycin A (Rot/AA, 1 µM), and 2-DG (50 mM). MIA PaCa-2 cells were suspended in DMEM (no sodium pyruvate, no glucose, Gibco #11966025) supplemented with 10% FBS and seeded in XF24 microplates at 1.6×104 cells/well, then incubated at 37°C in a 5% CO2 humidified incubator 24 h later, and cells were treated with indicated concentrations of glucose for 48 h. OCR values (pmol/min) and ECAR values (mpH/min) were normalized for protein (µg). (A) Representative traces of OCR values (pmol/min/µg) of MIA PaCa-2 cells cultured in DMEM medium without glucose (black trace) and in DMEM medium with 100 mM glucose (red trace). (B) OCR values (pmol/min) were normalized for protein (µg) and expressed as OCR (pmol/min/µg). In groups of Basal, Oligo, and FCCP, the OCR values were subtracted for Rot/AA. Data are expressed as mean ± SD. ** p < 0.01 vs. 0 mM, *** p < 0.001 vs. 0 mM, two-way ANOVA with Bonferroni post-hoc test. (C) oligomycin-sensitive respiration was expressed as mean ± SD. (D–F) ECAR values (mpH/min) were normalized for protein (µg). (D) Representative traces of ECAR values (mpH/min/µg) of MIA PaCa-2 cells cultured in DMEM medium without glucose (black trace) and in DMEM medium with 100 mM glucose (red trace). (E) ECAR values (mpH/min/µg) were subtracted for 2-DG and expressed as mean ± SD. ** p < 0.01 vs. 0 mM, *** p < 0.001 vs. 0 mM, two-way ANOVA with Bonferroni post-hoc test. (F) Ratio of basal OCR value and basal ECAR value. *** p < 0.001 vs. 0 mM, one-way ANOVA with Bonferroni post-hoc test. (G–I) MIA PaCa-2 cells were cultured in DMEM (no sodium pyruvate, no glucose, Gibco #11966025) supplemented with 10% FBS. (G) Representative blots of protein extracts of isolated mitochondria (mito) and cytosolic fraction (cyto) from MIA PaCa-2 cells treated by indicated concentrations of glucose for 48 h. (H) Representative immunofluorescence images (scale bar: 36.8 µm) of MIA PaCa-2 cells treated without (CTL) or with 200 mM glucose for 48 h. Cells were stained with anti-TOM20 (red) and anti-c-Myc (green). (I) MIA PaCa-2 cells were treated by indicated concentrations of glucose for 48 h and transfected with EV or plasmids carrying ATPIF1 for 24 h. Cells were collected and lysed for coIP and WB. The blots are representative of three independent experiments.

Fig 3: IF1 participates in enhanced glycolysis driven by c-Myc. (A, C) HEK293T cells were transfected with empty vector (EV) or plasmids carrying ATPIF1 and incubated for 24 h. In (A), cells were lysed for coIP and WB. In panel C, isolated mitochondria (mito) were lysed for coIP and WB. (B) HEK293T cells were lysed for coIP and WB. In negative control (NC), only Protein A/G Plus Agarose Beads and ATPIF1 antibody were incubated with coIP buffer at 4°C overnight. In IF1, Protein A/G Plus Agarose Beads and ATPIF1 antibody were incubated with cell lysate at 4°C overnight. PonS indicated the ponceau S staining of transferred membraned. (D–J) WT HeLa cells were transfected with EV or plasmids carrying c-Myc and incubated for 24 h. (D) Cells were collected from XF24 Cell Culture Microplates after Seahorse experiment and lysed for WB. OXPHOS (E–J) and glycolysis (H–J) activities were evaluated by Agilent Seahorse XFe24 Analyzer before and after the addition of oligomycin (Oligo, 2 µM), FCCP (0.25 µM), rotenone plus antimycin A (Rot/AA, 1 µM), and 2-DG (50 mM). OCR values (pmol/min) were normalized for protein (µg). (E) Representative traces of OCR values (pmol/min/µg) in HeLa cells transfected with EV (black trace) and plasmids carrying c-Myc (purple trace). (F) OCR values (pmol/min/µg) were subtracted for Rot/AA and expressed as mean ± SD. (G) Oligomycin-sensitive respiration was expressed as mean ± SD. (H, I) ECAR values (mpH/min) were normalized for protein (µg). (H) Representative traces of ECAR values (mpH/min/µg) in WT HeLa cells transfected with EV (black trace) and plasmids carrying c-Myc (purple trace). (I) ECAR values (mpH/min/µg) were subtracted for 2-DG and expressed as mean ± SD. * p < 0.05 vs. EV, *** p < 0.001 vs. EV, two-way ANOVA with Bonferroni post-hoc test. (J) Ratio of basal OCR value and basal ECAR value in WT HeLa cells. *** p < 0.001 vs. EV, one-way ANOVA with Bonferroni post-hoc test. (K–M) Glycolysis activities of ?ATPIF1 HeLa cells. (K, L) ECAR values (mpH/min) were normalized for protein (µg). (K) Representative traces of ECAR values (mpH/min/µg) in ?ATPIF1 HeLa cells transfected with EV (red trace) and plasmids carrying c-Myc (violet trace). (L) ECAR values (mpH/min/µg) were subtracted for 2-DG and expressed as mean ± SD. (M) Ratio of basal OCR value and basal ECAR value in ?ATPIF1 HeLa cells.

Fig 4: IF1 binds to PGC1a and inhibits mitochondrial oxidative respiration. (A) Representative immunofluorescence images (scale bar: 10 µm) of HEK293T cells stained with anti-IF1 (red) and anti-PGC1a (green), and co-labeled with DAPI (blue). (B) HEK293T cells were transfected with EV or plasmids carrying ATPIF1 and incubated for 24 h. Cells were lysed for coIP and WB. (C) HEK293T cells were lysed for coIP and WB. In NC, only Protein A/G Plus Agarose Beads and ATPIF1 antibody were incubated with coIP buffer at 4°C overnight. In IF1, Protein A/G Plus Agarose Beads and ATPIF1 antibody were incubated with cell lysate at 4°C overnight. PonS indicated the ponceau S staining of transferred membranes. (D–I) ?ATPIF1 HCT116 cells were transfected with plasmids carrying c-Myc (?ATPIF1+c-Myc) or PPARGC1A (encoding PGC1a) (?ATPIF1+PGC1a), and WT HCT116 cells were transfected with plasmid carrying PPARGC1A (WT+PGC1a), then co-cultured for 24 h. OXPHOS (D–I) and glycolysis (G–I) activities were evaluated by Agilent Seahorse XFe24 Analyzer before and after the addition of oligomycin (Oligo, 2 µM), FCCP (0.25 µM), rotenone plus antimycin A (Rot/AA, 1 µM), and 2-DG (50 mM). OCR values (pmol/min) were normalized for protein (µg). (D) Representative traces of OCR values (pmol/min/µg) in ?ATPIF1+c-Myc (purple trace), ?ATPIF1+PGC1a (blue trace), and WT+PGC1a (gray trace). (E) OCR values (pmol/min/µg) were subtracted for Rot/AA and expressed as mean ± SD. *** p < 0.001 vs. ?ATPIF1+PGC1a, two-way ANOVA with Bonferroni post-hoc test. (F) oligomycin-sensitive respiration was expressed as mean ± SD. *** p < 0.001 vs. ?ATPIF1+PGC1a, one-way ANOVA with Bonferroni post-hoc test. (G, H) ECAR values (mpH/min) were normalized for protein (µg). (G) Representative traces of ECAR values (mpH/min/µg) in ?ATPIF1+c-Myc (purple trace), ?ATPIF1+PGC1a (blue trace), and WT+PGC1a (gray trace). (H) ECAR values (mpH/min/µg) were subtracted for 2-DG and expressed as mean ± SD. *** p < 0.001 vs. ?ATPIF1+PGC1a, two-way ANOVA with Bonferroni post-hoc test. (I) Ratio of basal OCR value and basal ECAR value. ** p < 0.01 vs. ?ATPIF1+PGC1a, *** p < 0.001 vs. ?ATPIF1+PGC1a, one-way ANOVA with Bonferroni post-hoc test.

Fig 5: IF1 is required for the protective effect of hypoxia on c-Myc/PGC1a-induced cell death. (A, C–E) The effects of IF1/c-Myc/PGC1a overexpression and CoCl2-induced hypoxic environment on cell viability in WT and ?ATPIF1 HeLa cells revealed by MTT assay. Viable (%) was expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. EV or WT, two-way ANOVA with Bonferroni post-hoc test. (F) The images of WT and ?ATPIF1 HeLa cells with overexpression of IF1/c-Myc/PGC1a and CoCl2 treatment for 24 h. The figures are representative of at least three independent experiments. (B) Expression of IF1 in HeLa clonal cells after disruption of ATPIF1 gene with sgRNA-2 using the CRISPR/Cas9 technique. Colony 3 (number in bold) was used for MTT assay as shown in (A, C–F). (G) HCT116 WT and IF1 KO cells were transfected with EV or plasmids carrying ATPIF1 for IF1 overexpression (IF1 OE), then incubated for 24 h, followed by MitoSOX staining.