Modulating HUVEC Responses to Glucose Stress

Human umbilical vein endothelial cells (HUVECs; ATCC) were cultured with CC-3162 EGM-2 Bulletkit (Lonza, MD, United States). According to the instructions of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States) and opti-MEM reduced serum medium (GIBCO life technologies, Grand Island, NY, United States), the cells cultured for 24h in 12-well plates were treated with miR-195-5p inhibitor, miR-195-5p mimic, si-smad7, or corresponding controls (GenePharma). After 6h, opti-MEM reduced serum medium was replaced by complete cell culture medium with high concentration of glucose (HG: 25mmol/L, D-glucose, G5500, Sigma-Aldrich) and negative control (NG: 25mmol/L, L-glucose, G8644, Sigma-Aldrich) for 48h (Li et al., 2020 (link)). In order to further verify the regulatory effect of TGF-β1/smads pathway, HUVECs were stimulated using 3μmol/L TGF-β1R-I inhibitor LY-364947 (LY, # 54678S, CST, Beverly, MA, United States) for 24h (Che et al., 2020 (link)). The cell treatment process was shown in Supplementary Figure 2.