Hepatocyte growth factor (hgf)

Fifty micrograms of total cell lysate was separated on a 10% SDS-PAGE gel, transferred on PVDF membrane and blotted with antibodies. HGF, VEGFA, VEGFR2 and MMP2 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). ERK1/2, phospho-ERK1/2 and α-Tubulin were from Cell Signaling and Sigma, respectively. HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were used to identify the protein of interest. Each experiment was repeated at least two times. Secreted VEGFA and HGF was detected either in conditioned media (CM) of cultured cells or patient's sera using ELISA kits from Ray Biotech (Norcross, GA, USA).

Following the manufacturer’s instructions, the levels of total TGFβ1 (Legend Max, BioLegend, San Diego, CA, USA) and HGF (RayBiotech, Peachtree Corners, GA, USA) were quantified by ELISA in the CM from HCT116 and LS174T cells (siCTRL and siKRAS) as well as in the media from all conditions collected at the final timepoint of the experiment with the CCD-18Co fibroblasts.

The PDGF, EGF, HGF, and TGF-β1 concentrations of the platelet releasates were determined with human enzyme-linked immunosorbent assays according to the manufacturer’s instructions (PDGF, EGF; PeproTech, Rocky Hill, CT, USA, HGF; Ray Biotech, Norcross, GA, USA, TGF-β1; Thermo Fisher Scientific).