Immunoblotting

To test the protein expression, HUVEC and HMVE cells were lysed with cell lysis buffer (Cell Signaling Technology (CST), #9803) supplemented with protease inhibitor cocktail (Thermo #78442). Proteins were separated by centrifugation at 12,000 g for 10 min at 4°C and protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, USA). Total cellular proteins were separated in 4–15% SDS-PAGE, transferred on PVDF membrane followed by 1 h blocking in skim milk at room temperature (RT). Immunoblotting was performed using primary antibodies against ALKBH5 (Abcam, #195377), METTL3 (Abcam, # ab195352), AKT (CST, #2920), phospho-AKT (CST, #4060), eNOS (CST, #9572), phospho-eNOS (CST, #9571), VEGF-A (Abcam, #46154), SPHK1 (CST, #12071) and GAPDH (CST, #5174). All primary antibodies are used at 1:1000 dilutions, overnight at 4°C. Species-specific HRP-linked secondary antibodies were used at 1:2000 dilutions at RT for 1 h. Signals were detected using Odyssey® Fc Imaging System (LI-COR Biosciences). For quantitative and statistical analysis, the ImageJ (NIH) and GraphPad PRISM software was used, respectively.