Fig. 3
Exosomal circTMEM181 from HCC is internalized by macrophage and sponged miR-488-3p in macrophage. a Schematic diagram: CD8+ T cells isolated from human PBMCs were co-cultured with Huh-7circOE, Huh-7ctrl, or THP-1 or the supernatant from THP-1 and Huh-7OE co-culture medium. b Flow cytometry analysis was used to evaluate proliferation of CFSE-labeled CD8+ T cells in different conditions (Sup.: supernatant of THP-1 and Huh-7OE co-culture medium; one-way ANOVA: ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: not significant). c Flow cytometry analysis of PD1, TIM3, and TIGIT expression on CD8+ T cells from different culture conditions. (MFI, mean fluorescent intensity; one-way ANOVA: ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: not significant). d Representative picture of exosomes enriched using ultracentrifugation from medium of Huh-7 by transmission electron microscopy (Left); Nanoparticle tracking analysis was performed to analyze the size distribution of enriched exosomes from different HCC cell lines (Right). e Exosomal markers CD63 and TSG101 were detected on enriched exosomes across four human HCC cell lines and two mouse HCC cell lines. f CircTMEM181 expression was analyzed in cell lysates or extracellular vesicles (EVs) across different manipulations of various cell lines (OE: overexpressing circTMEM181; Sh: circTMEM181 knock down). g CircTMEM181 expression was analyzed in THP-1 macrophages co-cultured with or without Huh-7ctrl or Huh-7OE or GW4869 (one-way ANOVA: ***: p < 0.001, **: p < 0.01, ns: not significant). h Immunofluorescence shows exosomes pre-labeled with PKH-67 (green) from Huh-7OE can be internalized by THP-1 (white arrow). i RNA immunoprecipitation with circTMEM181-specific probes shows enrichment of RNAs in THP-1 overexpressing circTMEM181 (THP-1circOE) compared to the control (THP-1ctrl). j Schematic diagram: putative binding sites of wild-type circTMEM181, hsa-miR-488-3p, hsa-miR-1298 and mutant circTMEM181. k Detection of wild-type circTMEM181-labeled luciferase (WT) or mutant circTMEM181-labeled luciferase (MU) activity in HEK293T cells after miR-488-3p or miR-1298 transfection (t test, *: p < 0.05, **: p < 0.01, ns: not significant). l RNA immunoprecipitation with circTMEM181-specific probes performed in HEK293T cells using biotin-labeled miR-488-3p mimics and a negative control (NC). m CD8+ T cells isolated from human PBMCs were co-cultured with supernatant of medium from co-culturing Huh-7OE and THP-1 overexpressing miR-488-3p (THP-1miR−OE). n FISH analysis of circTMEM181 (green) and miR-488-3p (red) demonstrates their colocalization in the THP-1 cytoplasm