Fig. 2

Exosomal transfer of miR-3188 from fibroblasts to HNC cells. a HN4 and HN30 cells were culture with DiO-labeled exosomes from CAFs for 24 h, and Laser Scanning Confocal Microscopy was used to analyze the internalization of CAF-derived exosomes into HNC cells (Scale bar = 20 μm). b A mixture of CAL27 cells and CAFs expressing CD63-GFP fusion protein were used to generate xenografts in nude mice, and the mice were treated with or without GW4869 (2 mg/kg). Immunofluorescence staining was used to detect the stroma cell-derived exosomes internalization by HNC cells in xenografts (S, stroma; T, tumor; Scale bar = 20 μm). c Conditioned medium derived from CAFs transfected with cy3-labelled miR-3188 was added to HN30 cells for 24 h, and the red signal was detected using Confocal Microscopy (Scale bar = 25 μm). d Light microscopy and fluorescent images of HN30 cells after co-culture with CAFs loaded with cy3-miR-3188 for 24 h (Scale bar = 40 μm). e NF-CM or CAF-CM were treated with RNase A (2 mg/ml) or/and 0.1% Triton X-100, miRNA in CM was isolated and miR-3188 was detected using real-time PCR. f miR-3188 expression levels in CM, CM dislodged with exosomes and exosomes derived from CM of NFs and CAFs. g Primary cultured normal epithelial cells and cancer cells were treated with CM (Left) or exosomes (Right) derived from control cells (Ctrl CM, Ctrl Exos), NFs or CAFs for 48 h, and miR-3188 was detected using real-time PCR. h The miR-3188 expressions in original NFs or NFs co-cultured with cancer cells (Co-NFs) were detected. i The normal cells and cancer cells were respectively treated with exosomes derived from control cells, original NFs or NFs co-cultured with cancer cells (Co-NF-Exos). Then the miR-3188 expression levels were detected. j After HNC cells were treated with CM of HNC cells, CM of fibroblasts with different miR-3188 expression or CM dislodged with exosomes, miR-3188 expressions were detected using real-time PCR. (ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)