Fig. 4

GOLM1 increases exosomal PD-L1 levels from HCC cells. a Immunofluorescence staining showed a co-localization of GOLM1 with exosome marker CD63 (left panel) or PD-L1 (middle panel) and CD63 with PD-L1 (right panel) in the multivesicular endosomes (MVEs), which are the precursor forms of exosomes before released. b Co-IP determined the association of GOLM1 and exosome markers CD63, CD9, TSG101, and Alix from PLC cells transfected with Flag-GOLM1. c Co-IP demonstrated the association of exogenous GOLM1 with PDL-L1 in GOLM1^Flag PLC cells and exosomes: PLC cells transfected with FLAG-GOLM1 or empty vector (control) were subjected to IP using anti-FLAG magnetic beads. d The association of endogenous PD-L1 with GOLM1 and CD63 in MHCC-97H cells was validated. e The association of endogenous PD-L1 with GOLM1 in MHCC-97H exosomes was validated. f GST pulldown assays showed a direct interaction between GOLM1 and PD-L1. GST- GOLM1 was expressed in E. coli and purified. Full-length PD-L1 was produced via in vitro translation. g Mapping the regions of GOLM1 that interact with PD-L1. Various FLAG-tagged truncated GOLM1 were prepared to test the interaction with GST-PD-L1, GOLM1 was detected by anti-Flag antibody after GST pulldown. Coomassie blue staining of one-tenth of the amount of each GST-PD-L1 fragment. h Western blotting analysis showed after Rab27a was knocked down, intracellular accumulation of PD-L1 and GOLM1 was detected in MHCC-97H cells, whereas PD-L1 and GOLM1 in exosomes decreased significantly, and the abundance of exosomes, indicated by exosomal markers, ALIX, TSG101, CD63, and CD9, was inhibited significantly. i, j Western blot analysis of the exosomes isolated from an equivalent number (6 × 10⁷) of HCC cells indicating that GOLM1 facilitates the secretion of exosomal PD-L1. MHCC-97H cells with endogenous high-GOLM1 (i) and low-GOLM1 Hep3B cells (j) were used