Extended Data Fig. 6: An elevated OGT–FOXK2-SLC7A11 axis contributes to chemotherapy resistance in HCC, related to Fig. 7.
From: A ROS-mediated oxidation-O-GlcNAcylation cascade governs ferroptosis
a, FOXK2 KO Huh7 cells were reintroduced with either WT or FOXK2 S424A mutant. The cells were treated with increasing concentrations of 5-FU for 72 h, and cell viability was measured. n = 3 independent replicates. b, The indicated Huh7 cells (from a) were seeded and treated with 4 Gy irradiation. Colony-formation assays were conducted. n = 3 independent repeats. c. d, Huh7, Huh7 5-FUR, and Huh7 IRR cells were treated with increasing concentrations of 5-FU (c) or varying doses of irradiation (d) for 72 h, and cell viability was assessed. The IC50 values are indicated. n = 3 independent replicates. e, Intracellular ROS levels in HepG2 and 5-FU-resistant HepG2 (HepG2 5-FUR) cells were measured by flow cytometry. n = 3 independent experiments. f, The protein levels of SLC7A11, along with total O-GlcNAcylation, were determined in HepG2 5-FUR cells by immunoblotting with the specified antibodies (n = 3 experiments). g, FOXK2 O-GlcNAcylation in HepG2 5-FUR cells was analysed by the chemoenzymatic labelling method and determined by immunoblotting. h, SLC7A11 mRNA level in HepG2 5-FUR cells was determined by qPCR. n = 3 independent repeats. i, HepG2 and HepG2 5-FUR cells were treated with increasing concentrations of 5-FU for 72 h, and cell viability was assessed. The IC50 values are indicated. n = 3 independent replicates. j, Intracellular ROS levels in PLC/PRF/5 and 5-FU-resistant PLC/PRF/5 (PLC/PRF/5 5-FUR) cells were measured by flow cytometry. n = 3 independent experiments. k, The protein levels of SLC7A11 and total O-GlcNAcylation were determined in PLC/PRF/5 5-FUR cells by immunoblotting (n = 3 experiments). l, Schematic model illustrating a ROS-induced oxidation-O-GlcNAcylation cascade specific to ferroptosis, which contributes to drug resistance in HCC. Created in BioRender. Zhang, H. (2025) https://BioRender.com/ky9usx1. a,c,d,i, Data are presented as mean ± SEM, with statistical analysis performed using two-way ANOVA. b,e,h,j, Data are presented as the mean ± SEM; two-tailed Student’s t-test; ns, not significant.
