Fig. 3

VCP acts through downstream metabolite G3P. a Volcano plot representing metabolite changes in conditional medium from shVCP HCCLM3 cells compared with Control. b Schematic of G3P metabolism. c Extracellular G3P levels in shCtrl or shVCP HCCLM3 cells (n = 3). d G3P levels in fresh medium (FM) and conditional medium (CM) from HCCLM3 cells (n = 3). e G3P levels in interstitial fluid of spontaneous tumors (n = 3). f G3P levels in tumor interstitial fluid (TIF) and plasma from HCC patients (n = 31). g The proliferation of CD8⁺T cells co-cultured with shCtrl or shVcp Hepa1-6-OVA cells or treated with G3P using a Transwell system was measured by CCK8 assay (n = 3). Two-way ANOVA. h, i The percentage of cytokines production (h) and activation indicators (i) of CD8⁺T cells treated as in (g) were measured by flow cytometry (n = 3). j The proliferation of human CD8⁺T cells treated with G3P was measured by CFSE assay. k The proliferation of human CD8⁺T cells treated with G3P was measured by CCK8 assay (n = 3). Two-way ANOVA. l The percentage of Annexin v⁺ human CD8⁺T cells treated with G3P was determined by flow cytometry (n = 3). m The percentage of cytokines produced by human CD8⁺T cells treated with G3P was measured by flow cytometry (n = 3). n Secreted IFN-γ levels by human CD8⁺T cells treated with G3P. Analysis was performed after 24 h (n = 3). o The expression of activation indicators in human CD8⁺T cells treated with G3P was determined by flow cytometry (n = 3). p, q Representative images of tumor volumes from Hepa1-6-bearing C57BL/6 mice after intratumoral injection of PBS/G3P and sacrificed on day 25 (n = 5/group). Mean values ± SEM. Two-way ANOVA. Data are presented as mean values ± SD. Statistical significance was determined using two-sided t-tests, *P < 0.05, **P < 0.01, and ***P < 0.001