Fig. 2

Exosomes derived from isolated CAFs from CRC tissues contains miR-20a. A-C The identity of the CAFs was confirmed through the detection of the CAF surface markers Alpha Smooth Muscle Actin (α-SMA) and Fibroblast Activation Protein (FAP), as well as the fibroblast marker Vimentin, using RT-qPCR. The positive expression of α-SMA and FAP in the isolated and cultured cells strongly indicated the successful isolation of CAFs (p < 0.01), while higher levels of Vimentin were observed in NFs (p < 0.05). D The purified exosomes derived from CAFs and NFs displayed a spherical morphology, with their diameters falling within the range of 50 to 150 nm. This was verified through scanning electron microscopy (SEM) imaging. E Western blot analysis showed high expression of CD9 as an exosome-specific surface marker. The cytoplasmic protein marker Calnexin was present in the entire cell lysate but was absent in the isolated exosomes, suggesting that the exosome samples were free from contamination with other vesicles, such as those from the endoplasmic reticulum. The cropped blots are presented correspondingly. Full-length blots are presented in Supplementary Fig. 3. F Analysis using confocal microscopy unveiled the presence of red fluorescence of PKH26-labeled exosomes within the cytoplasm and perinuclear region of the recipient SW480 CRC cells, suggesting successful internalization of exosomes in CRC cells. The nuclear staining was carried out by DAPI. The bar represents 20 μm. G, H The expression levels of five candidate miRNAs in CAFs, NFs, and their corresponding exosomes. According to RT-qPCR results, miR-20a was found to be higher in CAFs (G) and their exosomes (H), when compared to NFs and their respective exosomes. All experiments were conducted in triplicate. NF Exo: NF-derived exosomes, CAF Exo: CAF-derived exosomes. Columns represent means of three different experiments; bars represent SD. *P < 0.05, **P < 0.01