Knockdown and Overexpression of PTBP1 in HUVECs

Human umbilical vein cells (HUVECs, PromoCell, C-12208) were cultured in endothelial cell growth medium (PromoCell, C-22010). To knockdown the expression of PTBP1, HUVECs were infected with lentivirus-shRNA-PTBP1 (LV-sh-PTBP1) (Gene Pharma, Inc.) at a multiplicity of infection (MOI) of 50 at 37 °C for 24 h, and then cultured with fresh medium for another 48 h at 37 °C in a 5% CO2 incubator before subsequent experiments. The cells infected with control lentivirus-shRNA (Gene Pharma, Inc.; MOI, 50) were used as a negative control. For the migration assay, HUVECs were incubated with 10 μg/mL mitomycin C (MedChemExpress) at 37 °C in a 5% CO2 atmosphere for 2 h^{50 (link)}. The LV-sh-PTBP1 sequences were as follows: 5’-CGGCACAGTGTTGAAGATCAT-3’. For the overexpression of ARRB1-L or ARRB1-S, HUVECs were infected with ARRB1-L overexpression lentivirus or ARRB1-S overexpression lentivirus. At 72 h after infection, the growth area was scratched. The cells were then photographed in five different fields at 0 h and 20 h after scratching using a Leica DMi8 microscope, respectively. Cell migration ability was calculated as a percentage of the wound area difference between 0 h and 20 h to the wound area at 0 h by ImageJ software.