Fig. 3: ZDHHC3 catalyzes B7-H4 palmitoylation at Cys130 impairing tumor immunity.

a Comparison of B7-H4-C130A palmitoylation with B7-H4-WT in 293T cells detected by ABE assay. b Immunoblot for B7-H4 WT and C130A in 293T cells treated with 2-BP for 24 h. c Representative immunoblot for B7-H4 WT and C130A and protein level quantification from three independent tests in 293T cells examined by cycloheximide (CHX) chase assay. Data are shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. d Normalized 23 human palmitoyltransferases (PATs) mRNA levels in MDA-MB-468, T-47D, SK-BR-3, and MCF7 cells using ACTB as reference. n = 4 per group. Data are shown as mean ± SEM. e Quantitative analysis of relative B7-H4 palmitoylation levels from three independent ABE screens by co-overexpressing the candidates PATs from (d) and B7-H4 in 293T cells. Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test to GST control. f Interaction between B7-H4-GFP and ZDHHC3-Flag in 293T cells. Immunoblot for B7-H4 in T-47D cells with ZDHHC3 overexpression (g) or knockdown (h) and in 4H11 cells with ZDHHC3 knockdown (i). shNT non-targeting shRNA. Zdhhc3 knockdown (shZdhhc3) and control (shNT non-targeting shRNA) 4H11 subcutaneous tumor growth (j), representative image (k). n = 6 per group. Subcutaneous implantation was conducted in 8-week-old female C57BL/6 mice. Data are shown as mean ± SEM (j). Statistical analysis was performed using two-way ANOVA (j). Flow cytometry analysis of the number (l) and percentage (m) of tumor-infiltrating CD8⁺ T cells, IFN-γ⁺CD8⁺ T cells (n) in subcutaneous 4H11 tumor-bearing mice. n = 6 per group. Subcutaneous implantation was conducted in 8-week-old female C57BL/6 mice. Data are shown as mean ± SEM. Statistical analyses were performed using two-tailed Student’s t test. Flow cytometry analysis of tumor-infiltrating TCF-1⁻TOX⁺ (o, p), and TCF-1⁺TOX⁻ (o, q) CD8⁺ T cells in subcutaneous 4H11 tumor-bearing mice. n = 6 per group. Subcutaneous implantation was conducted in 8-week-old female C57BL/6 mice. Data are shown as mean ± SEM (p, q). Statistical analyses were performed using two-tailed Student’s t test (p, q). Source data are provided as Source Data File.