Fig. 3: miR-375 is horizontally transferred from MCC cells to fibroblasts and induces a CAF-like phenotype.

MRC-5 cells (a, b, e, f, i, k, m) or primary skin fibroblasts (Fibro1.4) (c, d, g, h, j, l, n) were cultured in MCC conditioned medium (CM), in a Transwell chamber system (TC) or in direct cell-cell contact (DC) with MCC13, WaGa or PeTa cells. a- d The relative expression levels of miR-375 (a, c) and pri-miR-375 (b, d) in fibroblasts were determined by RT-qPCR. e–h The relative ACTA2, CXCL-2, and IL1B mRNA expression levels were determined in fibroblasts under TC conditions (e, g) or DC conditions (f, h) by RT-qPCR. i, j α-SMA protein expression in fibroblasts cocultured with MCC cells was determined by immunoblotting; β-tubulin served as loading control. IF staining of α-SMA (green) in MRC-5 (k) and Fibro1.4 cells (l) after direct coculture with MCC cells. Cellular membranes were stained with WGA (red); nuclei, with DAPI (blue). The graphs show the values of cell aspect ratio (length/ width) of MRC-5 (m) and Fibro1.4 (n) under indicated conditions (n = 50), presented as box-and-whisker plots. The scale bars represent 50 µm. For RT-qPCR experiments, Cq values were normalized to U6 or HPRT expression and compared to the ΔCq value of untreated MRC-5 or Fibro1.4 cells, respectively. Experiments were biologically replicated trice and were performed in triplicates. The error bars indicate the SDs; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.