Fig. 2. TIMP1^EV regulate TIMP1 levels in recipient fibroblasts.

A Immunoblot analysis of EV proteins CD63, CD81, ITGB1, CD9, TSG101, HSP90AA, TIMP1, and calreticulin as negative control in CM and EV fractions in 3 CRC cell lines. Whole-cell lysate (WCL) used as internal control. B Heat map of qPCR data illustrating differential expression pattern of various ECM genes in BJ and pF fibroblasts treated with CRC-EVs for 24 h. Values were normalised to CM controls. C TIMP1 mRNA levels in CM and EV treated fibroblasts (BJ and pF) (top) and corresponding TIMP1 protein levels (bottom). D Immunoblot of TIMP1 protein levels in BJ (top) and pF (Bottom) cells after 48 h CRC-EV and CM treatment and after a preceded heparin treatment (100 μg/ml) for 3 h. E Immunoblot showing TIMP1 expression in WCL and EV fraction derived from HCT116 TIMP1^KO clones (KO1 and KO2) (top) and HCT116 TIMP1^OE clones (OE1 and OE2) (bottom). Parental HCT116 cells (TIMP1^WT) used as reference. ACTB and EV markers ITGB1, Syntenin1, and CD63 were used as loading controls. F Immunoblot analysis of TIMP1 expression in pFs treated with CM and EVs from TIMP1^WT, TIMP KO1 and TIMP1 KO2 cells (top) and TIMP1^WT, TIMP OE1, and TIMP1 OE2 cells (bottom). Beta-actin (ACTB) was used as a loading control. G Densitometry quantification of TIMP1 protein expression normalised to ACTB shown in (G) using imageJ. All experiments have been performed at least 3 times. Error bars depict mean ± SEM. P values were calculated by unpaired t-test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.