Fig 2: Angptl2 deficiency accelerates T-cell-mediated tumor cell eradication by increasing IFN?-induced MHC-I expression in tumor cells. (A) Representative immunoblotting of ANGPTL2 in control KO (Control) and Angptl2 KO tRCC cell lines. Shown is a representative of two independent experiments. Hsc70 served as a loading control. (B) Relative expression of mRNAs encoding MHC-I (H2-K1 and H2-D1) and antigen presentation machinery-related molecules (B2m, Tap2, and Tapbp) in IFN?-treated or untreated control KO (Control) and Angptl2 KO (Angptl2 KO1 and KO2) cells (n = 4 per group). Levels in IFN?-untreated control cells were set to 1. Data are means ± SD. Statistical significance was determined by two-way ANOVA with Tukey's post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Representative histograms showing cell surface expression of H-2Kb and H-2Db in IFN?-treated or untreated control KO (Control) and Angptl2 KO (Angptl2 KO1 and KO2) cells. Shown is a representative of three independent experiments. (D) Representative histograms showing cell surface expression of SIINFEKL-bound H-2Kb in IFN?-treated or untreated Control-OVA, Angptl2 KO-OVA, and Angptl2 KO cells. Shown is a representative of three independent experiments. (E) Levels of indicated secreted T-cell cytokines (IFN? and TNFa) in culture supernatants of OT-I T cells, OT-I T cells co-cultured with Control-OVA, Angptl2 KO-OVA, or Angptl2 KO cells, and in corresponding tumor lines (n = 4 per group). Data are means ± SD. Statistical significance was determined by one-way ANOVA with Tukey's post hoc test. ***P < 0.001; n.s, not significant; n.d, not detected. (F) T-cell cytotoxicity assay with Control-OVA, Angptl2 KO-OVA, and Angptl2 KO cells (n = 6 per group). OT-I T cells were co-cultured 8 h with tumor cells at the indicated effector/target (E/T) ratios. Data are means ± SD. Statistical significance was determined by two-way ANOVA with Dunnett's post hoc test. **P < 0.01; ***P < 0.001.

Fig 3: ANGPTL2 promotes H3K27me3-mediated repression of MHC-I expression in tumor cells. (A) Relative expression of Irf1 and Nlrac5 mRNAs in IFN?-treated or untreated control KO (Control), Angptl2 KO, and Itga5 KO cells (n = 4 per group). Levels in IFN?-untreated control cells were set to 1. Data are means ± SD. Statistical significance was determined by two-way ANOVA with Tukey's post hoc test. **P < 0.01; ***P < 0.001; n.s, not significant. (B) Representative immunoblotting of phosphorylated STAT1 (Y701), total STAT1, and total IRF1 in control KO (Control), Angptl2 KO, and Itga5 KO cells treated with indicated concentrations of IFN?. Shown is a representative of three independent experiments. Hsc70 served as a loading control. (C) ChIP assay of H3K27me3 and H3K4me3 at the H2-K1 and Tap1 promoters in IFN?-treated control KO (Control), Angptl2 KO, and Itga5 KO cells (n = 4 per group). Data are means ± SD. H3K27me3 and H3K4me3 levels at each promoter are presented as a percentage of input. Statistical significance was determined by two-way ANOVA with Dunnett's post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Relative expression of Ezh2, Eed, Suz12, Mtf2, and Jarid2 mRNAs in control KO (Control), Angptl2 KO, and Itga5 KO cells (n = 4 per group). Levels in control cells were set to 1. Data are means ± SD. Statistical significance was determined by one-way ANOVA with Dunnett's post hoc test. ***P < 0.001; n.s, not significant. (E) Representative immunoblotting of EZH2 and JARID2 in control KO (Control), Angptl2 KO, and Itga5 KO cells. Hsc70 served as a loading control. Shown is a representative of three independent experiments. (F) ChIP assay of EZH2 at the H2-K1 and Tap1 promoters in IFN?-treated control KO (Control), Angptl2 KO, and Itga5 KO cells (n = 4 per group). Data are means ± SD. EZH2 occupancy of each promoter is presented as a percentage of input. Statistical significance was determined by two-way ANOVA with Dunnett's post hoc test. **P < 0.01.