Supplementary Figure 6 from Hypoxia-Induced Creatine Uptake Reprograms Metabolism to Antagonize PARP1-Mediated Cell Death and Facilitate Tumor Progression in Hepatocellular Carcinoma

Supplementary Figure 6. The USP10-PKLR axis serves as the key effector of creatine/SERPINE1-mediated Warburg effect and parthanatos antagonism. (A, B) Confocal microscopy (A) and cell fractionation (B) assays determining the subcellular localization of SERPINE1 in HCC cells. Scale bars, 10 μm. (C) Confocal microscopy analysis showing the colocalization of SERPINE1 and PKLR in HCC cells. Scale bars, 10 μm. (D) Endogenous co-IP assays confirmed the interaction between SERPINE1 and PKLR in HCC cells. (E, F) qRT-PCR was used to analyze the mRNA level of PKLR in SERPINE1-knockdown (E) and overexpression (F) cells. (G) Relative PKLR protein levels after CHX treatment. (H) Western blot analysis of the ubiquitination of PKLR upon SERPINE1 knockdown in HCC cells. (I, J) Endogenous co-IP assays confirmed the presence of the SERPINE1-PKLR-USP10 complex based on the LC-MS/MS data. (K) Confocal microscopy analysis showing the colocalization of PKLR and USP10 in HCC cells. Scale bars, 10 μm. (L, M) Endogenous and exogenous co-IP assays confirmed the interaction of PKLR and USP10 in HCC cells. (N, O) Western blotting analysis assessed the PARyaltion of USP10 in HCC cells upon MNNG or Olaparib administration. (P) Western blot analysis showing the degradation rate of USP10 in control and MNNG-treated groups. n = 3 independent experiments. Means ± SD are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significant difference.