Figure 2

Sub-lethal HHcy reduces endothelial migration and proliferation without suppressing VEGF/VEGFR transcripts and ROS level change. (A) Scratch wound assay in presence of Mitomycin C showing less migrated endothelial cells at 24 h post 2 mM Hcy treatment. (B) Quantification of migrated cells revealing that endothelial migration is significantly reduced in Hcy treated cells compared with control cells. (C) Bar plot of BrdU cell proliferation assay indicating that 2 mM Hcy treatment for 24 h causes proliferation defect in endothelial cells. (D) Representing scratch wound assay images depicting that as opposed to Hcy treatment, a similar concentration of Cys did not affect migration of endothelial cells when compared with untreated cells at 24 h. (E) Bar plot of measurement of migrated cells in scratch wound assay showing that contrary to Hcy treated cells, fold change in migrated cells is not altered upon 2 mM Cys treatment for 24 h compared with control cells. (F) BrdU cell proliferation assay revealing that unlike Hcy treated cells, 2 mM Cys treatment for 24 h did not influence endothelial proliferation. (G) Bars showing that compared with untreated cells, exposure to sub-lethal Hcy caused upregulation of mRNA levels of canonical VEGF signaling markers. 18S was used as internal control. (H) Bar graph showing that sub-lethal Hcy treatment does not induce ROS production in endothelial cells as determined by the fluorescent probe CM-H2DCFDA. For positive control, H₂O₂ was used. (I) Representative western blots showing protein levels of major antioxidant markers GPX1 and SOD1. Corresponding bar graphs showing densitometric analysis of the protein bands which suggest a non-significant but slight trend of upregulation of both the proteins in 2 mM Hcy (24 h) treated cells. As a loading control β-actin was used. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001 and ns is non-significant (p > 0.05).