Supplementary Figure 2 from Hypoxia-Induced Creatine Uptake Reprograms Metabolism to Antagonize PARP1-Mediated Cell Death and Facilitate Tumor Progression in Hepatocellular Carcinoma

Supplementary Figure 2. Creatine promotes HCC progression and parthanatos antagonism under hypoxia. (A-B) Cell proliferation and cell death of HCC control and SLC6A8-knockdown cells treated with MNNG were assessed by CCK-8 (A) and LDH leakage (B), respectively. (C) qRT-PCR was used to examine GATM and GAMT expression of HCC cells under normoxia or hypoxia. (D, E) Colony formation assays (D) and EdU incorporation (E) were used to determine the role of creatine in HCC cell proliferation and colony formation under hypoxia. Scale bars, 50 μm. (F) Cell migration and invasion were evaluated by transwell assays. Scale bars, 50 μm. (G, H) Cell apoptosis (G) and cell cycle (H) were measured using flow cytometry. (I, J) Immunofluorescence (I) and Western blotting (J) showed the nuclear translocation of AIF. Scale bars, 10 μm. (K) IHC staining of SLC6A8 expression in subcutaneous tumors. Scale bars, 50 μm. For cell experiments, n = 3 independent experiments; for animal experiments, n = 5 independent experiments. Means ± SD are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significant difference.