Supplementary Figure 5 from Hypoxia-Induced Creatine Uptake Reprograms Metabolism to Antagonize PARP1-Mediated Cell Death and Facilitate Tumor Progression in Hepatocellular Carcinoma

Supplementary Figure 5. The oncogenic role of creatine depends on SERPINE1 to form a creatine/SERPINE1/HIF-1α positive feedback loop. (A-C) CCK-8 (A), EdU incorporation (B) and colony formation assays (C) were used to determine the role of SERPINE1 in SLC6A8-knockdown cell proliferation and colony formation under hypoxia. Scale bars, 50 μm. (D) Western blot analysis of PAR polymer accumulation in hypoxic SLC6A8-knockdown cells with or without ectopic SERPINE1 expression. (E) IF analysis of AIF nuclear translocation in hypoxic SLC6A8-knockdown cells with or without ectopic SERPINE1 expression. Scale bars, 10 μm. (F, G) Intracellular pyruvate content and extracellular lactate content of hypoxic SLC6A8-knockdown cells with or without ectopic SERPINE1 expression. (H, I) The ECAR and OCR were measured in hypoxic SLC6A8 knockdown cells with or without ectopic SERPINE1 expression. (J) Representative confocal microscopy images showed the mitochondrial function in HCC cells treated with MitoTracker Green. Scale bars, 10 μm. (K) qRT-PCR analysis of the mtDNA content of hypoxic SLC6A8-knockdown cells with or without ectopic SERPINE1 expression. (L) Gross image and weight statistics of subcutaneous xenografts of HCC cells. (M) Western blot analysis of HIF-1α expression in hypoxic control and SLC6A8-knockdown cells upon creatine supplementation. (N, O) Relative expression of HIF-1α target genes in hypoxic HCC cells. (P, Q) Western blot analysis of HIF-1α expression in knockdown or ectopic expression of SERPINE1 HCC cells under hypoxia. For cell experiments, n = 3 independent experiments; for animal experiments, n = 6 independent experiments. Means ± SD are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significant difference.