Heparin sodium salt

HUVECs were purchased from ATCC and were grown in MCDB-131-complete media overnight. HUVECs for basal condition collection were grown in growth media of M199, 20% FBS, 0.05 g heparin sodium salt (Cat#9041-08-1, Alfa Aesar), and 15 mg ECGS (Cat#02-102, Millipore Sigma). Cells were routinely cultured on 1% of gelatin-coated plates at 37°C at 5% CO₂. For insulin stimulation (Cat#SLBW8931), cells were serum starved for at least 13 hr in 1% FBS (Atlanta Bio selected, Cat#S11110) or without FBS using endothelial cells growth basal medium (Lonza Cat#cc-3121) instead of MCDB-131-complete media. Insulin stimulation was used for the times indicated at 100 nM.

QuantiTect primer assays for α-SMA, vimentin, S100A4, Snail, Slug, Twist and glyceraldehyde 3-phosphate dehydrogenase were from Qiagen (Valencia, CA, USA). E-cadherin monoclonal antibody for immunofluorescence studies), β-catenin polyclonal antibodies (for immunofluorescence studies) and α-SMA polyclonal antibodies were from AbCam (Cambridge, MA, USA). Alexa 488-conjugated goat anti-mouse and Alexa 594-conjugated goat anti-rabbit secondary antibodies were from Life Technologies (Carlsbad, CA, USA). E-cadherin monoclonal antibody (for immunoblotting studies), β-catenin monoclonal antibody (for immunoblotting studies) and horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were from Cell Signaling Technologies (Danvers, MA, USA). Horseradish peroxidase-linked goat anti-mouse secondary antibodies were from GE Healthcare (Pittsburgh, PA, USA). Col IV was from BD Biosciences (San Jose, CA, USA). Uncoated and Matrigel-coated Transwell plates and heparin sodium salt were from Fisher Scientific (Waltham, MA, USA).