Fig 2: Influence of metformin on HIF-1 pathway activation induced by hypoxia, anoxia and cobalt chloride. a Influence of metformin on HIF-1 alpha stabilization. Upper panel: Multiple Myeloma cell lines were cultured for 24 h in different conditions in the presence and absence of metformin (5 mM) and subsequently HIF-1 alpha subunits accumulation was verified using the Western Blot. β-actin is shown as an internal control for equal loading. Lower panels: densitometry analysis of Western blot bands intensity normalized to β-actin. Each relative densitometry value is the average from three independent experiments. The mean ± SEM is shown. Student t test was used to evaluate influence of metformin on HIF-1 alpha stabilization. *p < 0.05 by student’s t test, p value between 0.05 and 0.1, by student’s t test was given as an indication of the trend. In all presented Western blot 30 µg of protein lysate was applied. b Influence of metformin on HIF-1 transcriptional response. Left panel: multiple myeloma cell lines, L363 and RPMI 8226, were cultured for 24 h in different conditions in the presence and absence of metformin (5 mM) and the influence of metformin on HIF-1 transcriptional response was assessed by RT-PCR. HPRT1 was used as an internal control for equal loading. Right panel: densitometry analysis of RT-PCR bands intensity normalized to HPRT1. Each relative densitometry value is the average from three independent experiments. The mean ± SEM is shown. Student t test was used to evaluate influence of metformin on HIF-1 triggered gene expression. *p < 0.05 by student’s t test, **p < 0.01 by student’s t test, p value between 0.05 and 0.1, by student’s t test, was given as an indication of the trend

Fig 3: Hypoxia contributes to the progression of CRC and upregulates circCCDC66 expression. (A) HIF1A mRNA and protein levels were measured in hypoxia-exposed HCT116 and SW620 cells by RT-qPCR and western blot analysis. *P<0.05 vs. normoxia. (B) RT-qPCR showed circCCDC66 expression in HCT116 and SW620 cells after hypoxia treatment. *P<0.05 vs. normoxia. (C) The viability of hypoxia-exposed HCT116 and SW620 cells was estimated by CCK-8 assay. *P<0.05 vs. normoxia. (D) Transwell assay was performed to detect cell invasion in hypoxia-exposed HCT116 and SW620 cells. (E) The migration ability of hypoxia-exposed HCT116 and SW620 cells was tested by wound healing assay. (F) The apoptosis rate of hypoxia-exposed HCT116 and SW620 cells analyzed by flow cytometry. *P<0.05 vs. normoxia. (G) The protein levels of Bax and Bcl-2 in hypoxia-exposed CRC cells were detected by western blot analysis. CRC, colorectal cancer; HIF1A, hypoxia inducible factor 1 subunit α.

Fig 4: Aerobic training and Hif1-α inhibitor effects on mouse breast tumor growthA. Western blot of Hif1-α in mouse tumors treated with Digoxin (+) or Saline (-). Hif1-α western blot in C3(1)SV40Tag-p16-luc mouse tumors: Mean densitometry normalized to β-actin: Control plus Digoxin(+) 1.87; Control plus saline (-) 4.50; Aerobic Training plus saline (-) 6.87; Aerobic Training plus Digoxin (+) 2.58; p = 0.01 for Aerobic Training group with Digoxin compared to Aerobic Training plus saline, all other groups compared p > 0.05. B. C3(1)SV40Tag-p16-luc aerobic training tumor study ± Digoxin. Tumor bearing mice were treated 5 days/wk. with aerobic training or control ± Digoxin (2mg/kg) or equivalent volume saline based upon total bodyweight. Values are log (tumor volume) (n = 6/group); ANOVA analysis in table (Right).

Fig 5: Bioinformatics analysis and the correlation between HIF-1α and FoxM1 in PCa. (A) High and low expression of HIF1A TCGA samples were used to identify DEGs. The results of GO and pathway enrichment applied on top 200 upregulated DEGs are shown in the bar plot, the higher the column and the darker the color, the smaller the P-value, and the corresponding enrichment entry is marked on the right. (B) The relative expression difference of DEGs was shown in the heatmap, where the green and red represented low and high expression, respectively. DEGs, differentially expressed genes; GO, Gene Ontology; FoxM1, Forkhead box M1; HIF1A, hypoxia-inducible factor 1α; has, homo sapiens; PCa, prostate cancer; PRAD, prostate adenocarcinoma; TCGA, The Cancer Genome Atlas. (C) Pearson correlation analysis of HIF1A and FoxM1 in TCGA prostate samples was performed; the red line represented the expression trend of the two genes. (D) The expression of FoxM1 in PRAD and normal prostate tissues (left panel), and the relationship between FoxM1 and Gleason scores (right panel). DEGs, differentially expressed genes; GO, Gene Ontology; FoxM1, Forkhead box M1; HIF1A, hypoxia-inducible factor 1α; has, homo sapiens; PCa, prostate cancer; PRAD, prostate adenocarcinoma; TCGA, The Cancer Genome Atlas.