Genipin

Human HK-2 renal proximal tubular epithelial cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin. miR-214-5p (100 ng; 5′-GGCCTGGCTGGACAGAGTTG-3′) and negative control (100 ng; 5′-CCCCCCCCCCCCC-3′) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). miR-214 mimics and mimic-negative control were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). Following 48 h of transfection, cells were cultured in DMEM supplemented with 35 mM glucose for 24 h for further analysis. Cells were then treated with 2 nM MK 2206 dihydrochloride (Akt inhibitor; MedChemExpress, Shanghai, China) or UCP2 inhibitor (genipin; 50 µM; MedChemExpress, Monmouth Junction, NJ, USA) for 44 h and cultured in DMEM supplemented with 35 mM of glucose for 24 h for further analysis.

Primary human retinal microvascular endothelial cells (HRECs, three to four passages; Cell Systems, Kirkland, WA, USA) were cultured in Endothelial Cell Medium (ScienCell, Carlsbad, CA, USA) containing 5% fetal bovine serum, 1% endothelial cell growth supplement, 100 U/mL penicillin, and 100 µg/mL streptomycin at a 5.5-mM d-glucose concentration for normal glucose (NG) and 30-mM for high glucose (HG). HRECs cultured in 5.5-mM d-glucose and 24.5-mM mannitol were used as an osmotic control.
Adenoviruses (adenovirus serotype 5) Ad-shUCP2 (expressing short hairpin RNA [shRNA] targeting human UCP2; GenBank NM_003355.3), Ad-UCP2 (overexpressing human UCP2 mRNA), vehicle control Ad-sh-ctrl (Ad-U6-CMV-MCS), and Ad-ctrl (Ad-CMV-MCS-HA) were purchased from OBiO Technology (Shanghai, China). The knockdown target sequences are available in Supplementary Table S1. Endothelial cells were infected with Ad-UCP2 or Ad-shUCP2 at a multiplicity of infection of 10 or 150, respectively, or with corresponding vehicle control at approximately 70% confluence. Inhibitors for SIRT3 (3-TYP, 16 µM; MedChemExpress, Monmouth Junction, NJ, USA) and UCP2 (genipin, 20 µM; MedChemExpress) were used individually or in combination with the adenoviruses.