Fig. 3 | Nature Communications

Fig. 3

From: Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin

Fig. 3

Replacement of endogenous HK2 with WT and HK2 mutants, and GCK. a Immunoblot analysis of engineered Huh7 cell lines for both endogenous HK2 KD and overexpression of different hexokinase isoforms. HA-tagged WT rat HK2 and mutants, resistant to silencing by human shRNA, and HA-tagged GCK were stably expressed in Huh7 cells expressing Dox-inducible HK2 shRNA. EV—empty vector, MTD—mitochondrial binding deficient mutant, SA—kinase dead mutant. b Analysis of in vitro hexokinase activity in the engineered stable cell lines. c Relative BrdU incorporation assay in the engineered cell lines. d AIG analysis in engineered cell lines. e, f Seahorse metabolic analysis (ECAR) of control cells (shNt EV), HK2 KD cells (shHK2 EV), shHK2 EV cells expressing WT HK2 (shHK2 HK2), expressing mitochondrial binding deficient mutant (shHK2 MTD), or glucokinase (shHK2 GCK). The cells were exposed to Dox for 3 days prior to analysis. The results are presented as the mean ± SEM; **p < 0.01 vs. Nt EV $p > 0.07 vs. EV—Dox; #p < 0.05, and ##p < 0.01 vs. EV + Dox, by Student’s t-test. Experiments were performed at least twice in triplicates

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