Figure 4. CAFs and NOFs are biochemically and morphologically different and CAF exosomes can also be transferred to CRC cells. (A) Western blot of paired primary NOFs and CAFs for myofibroblastic markers alpha-smooth muscle actin (α-SMA), fibronectin ED-A (ED-A FN1), palladin and vimentin. HSC-70 was used as an equal loading control. (B) Light microscopy of representative primary NOF and CAF cells (10x). (C) Fluorescence microscopy demonstrating phalloidin staining of F-actin filaments (green), counterstained with DAPI (blue; 40x). (D) Mean surface area and (E) intensity of phalloidin staining in a representative NOF-CAF pair. (F) Flow cytometry of DLD1 cells (control) and DLD1 cells co-cultured with CAF exosomes (exosome). The proportion of cells under the M1 region is given as a percentage. (G) Co-culture of CAF exosomes with DLD1 and SW480 cells with resultant increase in miR-199b and miR-21-5p. Data is presented as mean +/- SEM. Student’s t-test (D, E) or paired t-test (F, G): * p<0.05, ** p<0.01, *** p<0.001.