Cell lines

15×10⁴ Huh7 cells were transduced for GCK expression at different multiplicities of infection (lentiviral transduction using the pLEX-GCK construct). The Huh7-GCK⁺/HK2⁺ cells were then cultured for 7 days with puromycin (1 μg/mL) before amplification. HK2 knock-out was achieved using the CRISPR/Cas9 system as previously described⁵⁶ to obtain Huh7-GCK⁺/HK2⁻ cells. Briefly, a single guide RNA (sgRNA) pair was designed for double nicking using the CRISPR Design Tool (http://tools.genome-engineering.org). The guide sequence oligos (sgRNA₁(HK2): 5’-CACCGTGACCACATTGCCGAATGCC-3’ and sgRNA₂(HK2): 5’-CACCGTTACCTCGTCTAGTTTAGTC-3’) were cloned into a plasmid containing sequences for Cas9 expression and the sgRNA scaffold (pSpCas9(BB)-2A-GFP, Addgene plasmid #48138). 48 h post-transfection, cells were sorted by FACS based on the transient expression of GFP and cloned by limiting dilution. Effective deletion of HK2 was assessed by qPCR.

For HK2 knock-down, Huh7-GCK⁺/HK2⁺ cells were transduced with lentiviral vectors expressing HK2-targeting shRNAs, and antibiotic selection was applied (hygromycin; 100 µg/ml). The HK2-targeting sequence 5’-CCGGCCAGAAGACATTAGAGCATCTCTCGAGAGATGCTCTAATGTCTTCTGGTTTTTT-3’ was cloned in the pLKO.1 hygro vector (a gift from Bob Weinberg; Addgene plasmid #24150). HK2 expression in Huh7-GCK⁺/HK2⁺ and Huh7-GCK⁺/HK2⁻Sh was analyzed on cell lysates by western blotting (Supplementary Fig. 10).