Supplementary Figure 3 from Hypoxia-Induced Creatine Uptake Reprograms Metabolism to Antagonize PARP1-Mediated Cell Death and Facilitate Tumor Progression in Hepatocellular Carcinoma

Supplementary Figure 3. Creatine antagonizes parthanatos by enhancing the metabolic reprogramming of hypoxic HCC cells. (A) Heatmap of the DEGs in hypoxic Huh7 SLC6A8-knockdown cells compared with control cells. (B-C) KEGG and GO analyses of Huh7 RNA-seq data. (D) Gene set enrichment analysis of Huh7 cells. The normalized enrichment score (NES) was shown. (E, F) 2-NBDG absorbance (E) and glucose uptake (F) of hypoxic HCC cells were measured upon creatine supplementation. (G) HK and PK activity of hypoxic HCC cells were measured. (H, I) The ECAR (H) and OCR (I) were measured upon creatine supplementation in hypoxic HCC cells using a XF Extracellular Flux Analyzer. (J) ROS of hypoxic HCC cells was measured using flow cytometry. (K, L) The MDA level (K) and GSH/GSSG ration (L) of hypoxic HCC cells were measured. (M, N) The ECAR of HCC cells with or without MNNG under normoxia or hypoxia were measured. (O) Western blot examined the PAR polymer accumulation in hypoxic HCC cells with or without 2-DG (10mM) administration. (P) Western blot showed the phosphorylation levels of Smad2 and Smad3 in hypoxic HCC cells upon creatine supplementation. (Q, R) Western blot analysis was performed to evaluate the effects of SB525334 (Q) and AZ3146 (R) on Smad2/3 phosphorylation. (S) Western blot showed the knockdown efficiency of MPS1. (T) Western blot showed the Smad2/3 phosphorylation of hypoxic HCC cells with MPS1-knockdown rescued by wild-type or mutant MPS1 in the presence or absence of creatine treatment. (U) IF and western blot analyses examined the nuclear translocation of Smad4 in hypoxic HCC cells. n = 3 independent experiments. Means ± SD are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significant difference.