a qRT-PCR assay revealed the influence of WSX1 on PD-L1 mRNA expression level (n = 3 independent experiments). b Influence of WSX1 on the expression of exogenous PD-L1-FLAG. SNU449 and SNU475 cells were transfected with plasmid DNA encoding PD-L1-FLAG with or without WSX1 co-transfection and analyzed by Western blotting. c Immunoblot analysis of PD-L1 protein levels after treatment with proteasome inhibitor MG132 for 12 h. d Effect of MG132 on cell surface PD-L1 expression examined by flow cytometry. MG132 inhibited WSX1-mediated PD-L1 reduction in both SNU449 (n = 5 independent experiments, P = 0.0027) and SNU475 cells (P = 0.0362). e Influence of WSX1 on PD-L1 ubiquitination. Cells were pretreated with MG132 for 12 h, and cellular PD-L1 protein was pulled down by specific PD-L1 antibodies. PD-L1 ubiquitination was analyzed by anti-ubiquitin antibodies. f Impact of WSX1 on half-life of PD-L1 protein in HCC cells. HCC cells were treated with 25 mM CHX for 0, 4, 8, and 12 h, and cell lysates were collected separately and analyzed for PD-L1 protein levels. g Influence of WSX1 on protein levels of p-GSK3βSer9, total GSK3β, p-β-cateninSer33/Ser37/Thr41, and total β-catenin. β-catenin was directly phosphorylated by GSK3β at Ser33/Ser37/Thr41, and the level of p-β-cateninSer33/Ser37/Thr41 indirectly reflects GSK3β activity. h Influence of GSK3β inhibitor LiCl or GSK3β knockdown on PD-L1 expression. Cell surface PD-L1 expression on WSX1-overexpressing 449WSX1 and 475 WSX1 cells was analyzed by flow cytometry after treatment of LiCl or transfection of GSK3β shRNAs for 48 h. i Statistical analysis results showing that both LiCl treatment (P = 0.0135 in 449WSX1 and P = 0.0052 in 475WSX1) and GSK3β knockdown (P = 0.0020 in 449WSX1 and P = 0.0015 in 475WSX1) increased the proportion of PD-L1+ HCC cells (n = 5 independent experiments). j Schematic of site-directed mutation of the consensus motif on PD-L1-NP (S176A, T180A, and S184A), which could not be phosphorylated by GSK3β. k SNU449 and SNU475 cells transfected with PD-L1-wild type (WT) or PD-L1-NP (mutated) alone or co-transfected with WSX1. Alterations in cell surface PD-L1 expression levels were determined by flow cytometry. l Quantification of PD-L1 MFI ratio. Site-directed mutation in PD-L1-NP impaired WSX1-mediated PD-L1 downregulation in both SNU449 (n = 3 independent experiments, P = 0.0066 compared to PD-L1-WT) and SNU475 cells (P = 0.0004 compared to PD-L1-WT). Quantitative data are presented as mean ± SD and analyzed by one-way ANOVA or Student t test. Tukey-Kramer multiple comparison test was used for pairwise comparisons in the ANOVA analysis. Unless otherwise noted, data and images shown are representative of 3 independent experiments. All statistical tests were two-sided. *P < 0.05, **P < 0.01, ***P < 0.001. CHX cycloheximide, SP signal peptide, TM transmembrane domain, ECD extracellular domain, ICD intracellular domain, MFI mean fluorescence intensity. The gating strategy for sorting PD-L1+ HCC cells is shown in Supplementary Fig. 8b. Source data are provided as a Source Data file.