Rhplgf

The structure of fibrillary actin (F-actin) was determined by TRITC-phalloidin (Sigma Chemical, St Louis, MO, USA) staining using HLMECs seeded on LabTek-II chamber slides (Fisher Scientific, Pittsburgh, PA, USA) at a density of 5 × 10⁴. The cells were cultured overnight and then stimulated for 1 h with different concentrations of rhPlGF (R&D Systems, Minneapolis, USA). They were then washed with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized for 5 min with 0.5% Triton X-100 (Sigma Chemical, St Louis, MO, USA). Staining with TRITC-phalloidin (100 nM) for 30 min in the dark was followed by counterstaining for DNA with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma Chemical, St Louis, MO, USA). F-actin was detected with a Leica-TCS-SP confocal microscope and the accompanying software (Leica v2.6.1).

The cell proliferation assay was carried out in a 96-well plate using cells seeded at a density of 1 × 10⁴/well and treated for 48 h with different doses of recombinant human PlGF (rhPlGF, R&D Systems, Minneapolis, USA). A CCK-8 assay (CCK-8, Dojindo, Japan) to determine cell proliferation was performed according to the manufacturer’s instructions. HLMEC migration was assessed in a ‘scratch-wound’ assay22 (link).