Fig 2: Change in mitochondrial dynamics and biogenesis protein content in the skeletal muscle of control and LP offspring: (A) representative Western blots of mitochondrial dynamics and biogenesis genes tested in controls (C) and low-protein (LP) group; (B–K) depicts relative expression of different genes: (B) Opa1; (C) Mfn1; (D) Mfn2; (E) Fis1; (F) Pgc1b; (G) Nrf1; (H) Erra; (I) Vdac1; (J) Cox 4; (K) Sirt1. The blot density was calculated by densitometry scanning and normalized to GAPDH. Data represent mean ± SEM (* p < 0.05, ** p < 0.01); n = 8.

Fig 3: NRF2 negatively regulate the expression of ERR1(A) The relationship between NRF2 and ERR1 mRNA expression was retrieved from TCGA dataset using www.cbioportal.org and the correlation was analyzed by Pearson correlation and Spearman correlation. (B, C) The expression of ERR1 mRNA and protein levels was measured by qRT-PCR (B) and immunoblotting (C) in MCF7 cells. (D, E) The expression of ERR1 mRNA and protein levels was measured by qRT-PCR (D) and immunoblotting (E), respectively, in MDA-MB-231 cells. All data are means of three experiments, bar: SD, *P < 0.05; **P < 0.01; ***P < 0.005. (F, G) Binding of NRF2 to the ERR1 or HO-1 promoter in MCF7 (F) and MDA-MB-231 cells (G). Anti-NRF2- or IgG-immunoprecipitated chromatin was amplified using the indicated primer pairs. Results obtained by real-time PCR are expressed relative to amplified input. n = 3. Conventional PCR products from duplicate ChIP were analyzed on agarose gels. Input was diluted 1/100 before PCR. HO-1 gene served as positive control.

Fig 4: ERRα modulates cancer cell proliferation and metabolism. (A) WST-8 and BrdU assays were performed to examine the viability and proliferation capacity of PANC-1 and MDA-MB-231 cells after modulation of ESRRA levels by siRNA and expression plasmid. The data are the mean ± SD (n = 5). (B) After transfection with the ERRα expression plasmid and siRNA ERRα, cells were cultured for an additional two weeks and stained with Diff-Quik to determine colony formation capacity. (C) The cell cycle was determined by flow cytometry after the transfection of ERRα siRNA. The protein levels of cell cycle-related proteins, such as cyclin A/D/E were determined by immunoblot analysis after transfection with ERRα siRNA. Data are mean ± SD (n = 3). (D) ATP production in both high ERRα and low ERRα cells was detected. Data are mean ± SD (n = 5). (E) The intracellular accumulation of ROS in two cell lines was examined following the depletion of ERRα. Data are mean ± SD (n = 5). (F) Expression of mitochondriogenesis-related mRNAs NRF1, PGC-1A, TFAM, and CYBA. Data are mean ± SD (n = 6). (G) A glycolysis inhibition test with 2-deoxyglucose and a glucose deprivation test with glucose were performed to measure the anti-metabolic stress ability of tumor cells. Data are mean ± SD (n = 5).

Fig 5: Expression of genes involved in mitochondrial dynamics and biogenesis in the skeletal muscle of control and LP offspring. qPCR analysis was conducted to determine expression of mitochondrial dynamic genes: (A) Opa1; (B) Mfn1; (C) Mfn2; (D) Drp1; (E) Fis1; and biogenesis genes: (F) Pgc1b; (G) Erra; (H) Nrf1; (I) Nrf2; (J) Sirt1; (K) Vdac1; (L) Cox 4b. The mRNA expression was normalized relative to Cyclophilin A expression. Data represent mean ± SEM (* p < 0.05, ** p < 0.01); n = 8.