Trolox

BMVEC alone or in co-culture with pericytes were plated on collagen type I coated 96W20idf electrode arrays (Applied Biophysics, Troy, NY) and were treated with 25 mM glucose or AGEs for 30 hr. To inhibit RhoA or Rac1 GTPase activity, cells were pretreated with specific inhibitors, 1 μg/ml CT04 (Cytoskeleton) or 75 μM NSC23766 (EMD-Millipore, Burlington, MA), respectively. To inhibit ROS or apoptosis, cells were pretreated with specific inhibitors, 25 μM Trolox or 100 μM Z-VAD-FMK (both from Selleck Chemicals), respectively. TEER measurements were performed using the 1600R ECIS System (Applied Biophysics, Troy, NY) as described^{21 (link)–23 (link)}. The results are presented as the average percent change from baseline TEER (expressed as average ± SEM) from at least three independent experiments consisting of four to six replicates each.

BPTES (HY-12683), CBR-5884 (HY-100012), SHIN1 (HY-112066), DS18561882 (HY-130251), and sorafenib (HY-10201) were purchased from MedChemExpress. Trolox (S3665), lenvatinib (S1164) were from selleckchem. Crystal violet (V5265), H₂DCFDA (D399), MitoSOX^TM Red (M36008), monobromobimane (mBBr, M1378), Propidium Iodide (P3566), RNase A (10109169001), (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT, M6494), Hoechst 33342 (H3570), and Lipofectamine^TM RNAiMAX Transfection Reagent (13778150) were obtained from Thermo Fisher Scientific. H₂O₂ (216763) was purchased from Sigma-Aldrich. Triton X-100 (0694) was purchased from AMRESCO. Primary antibodies against SHMT2 (sc-390641), PHGDH (sc-100317), and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology. MTHFD2 (98116 S) was acquired from Cell Signaling Technology. Secondary antibodies of the mouse and rabbit were from Cell Signaling Technology and Abcam respectively (7076 S and ab6721). Small interfering RNA (siCTRL and siATF4) oligonucleotides were purchased from Santa Cruz Biotechnology (sc-37007 and sc-35112, respectively).