Chemicals and reagents
Primary antibodies against phospho-MLC (3674), MLC (8505), phospho-mTOR (2971), mTOR (2983), α-tubulin (3873), GAPDH (2118), phospho-RPS6 (4585), and RPS6 (2217); the RhoA Activation Assay Kit (8789); and rapamycin (8789) were purchased from Cell Signaling Technology (Danvers, MA, USA). FITC-Phalloidin (P5282) and Y-27632 (S1049) were purchased from Sigma Aldrich (Saint Louis, MO, USA) and Med Chem Express (Newark, NJ, USA), respectively.
Cell culture and glucose treatment
HRGECs (PS-4000) were provided by ScienCell Research Laboratories (Kirkland, WA, USA). HRGECs were maintained in endothelial cell medium with 1% endothelial cell growth supplement and 10% serum (ScienCell Research Laboratories). HRGECs were pretreated with or without rapamycin for 2 h and then stimulated with either 5.5 mM glucose (normal glucose; NG) or 30 mM glucose (high glucose; HG) (R&D Systems) for 16 h before western blotting or immunofluorescence analysis.
Western blot analysis
Western blotting was performed as described previously . Briefly, HRGECs were lysed with lysis buffer containing proteinase and phosphatase inhibitors (Roche, USA). Protein concentration was measured via BCA assay (Beyotime, Beijing, China). The same amount of protein was isolated using SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk and incubated with primary antibody overnight at 4 °C. Then, the membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at 20–24 ℃. Protein bands were detected using ECL Super Signal reagent (Pierce, 34078) and visualized using a digital gel image analysis system (BIO RAD, Hercules, CA, USA).
F-actin staining assay
For the F-actin stress fiber immunofluorescence assay, confluent HRGEC monolayers were grown on glass coverslips precoated with 0.1% gelatin. After treatment, the cells were fixed with 4% paraformaldehyde for 10 min and then blocked with PBS containing 1% bovine serum albumin. Next, the cells were incubated with FITC-Phalloidin for 1 h and stained with DAPI. Confocal images were acquired with a laser-scanning confocal microscope (FV1000-IX81, Olympus). Image analysis was performed using FV10-ASW Viewer software (Ver 4.1, Olympus Life Science, Japan).
Transendothelial electrical resistance (TEER) assay
HRGECs were seeded on transwell inserts (0.4 µm pore, Millipore, USA) and grown to confluence. The TEER of the HRGEC monolayer was measured using a Millicell-ERS voltohmmeter (Millipore, Burlington, MA, USA). Resistance values of the experimental groups are shown in units of Ω cm2. The transwell TEER of each group was recorded and normalized by subtracting the baseline TEER.
Transendothelial albumin permeability assay
HRGECs were grown to confluence on a 0.4 μm pore transwell insert (3413, Coring). After treated with NG or HG with or without inhibitors for 24 h, medium containing horseradish peroxidase (HRP)-labeled albumin (50 μg/mL; Solarbio, Beijing, China) was added to the top chamber. The concentration of albumin in the chambers was measured using the TMB Soluble Substrate kit (Solarbio, China), and absorbance values were recorded using a microplate reader (Elx 800, BioTek). The permeability coefficient of albumin (Pa) was calculated using the equation Pa = [A]/t × 1/A × V/[L], where [A] and [L] represent the albumin concentration in the bottom and top chambers, respectively; t represents time (s), A represents the area of the membrane (cm2), and V represents the volume of the bottom chamber (uL).
Rho activity assay
Rho activity in HRGECs was analyzed using the Active Rho Detection Kit. Briefly, HRGECs grown in 100 mm petri dishes were treated with either 5.5 or 30 mM glucose and/or pretreated with rapamycin (100 nM, 1 h). Next, the cells were lysed with cell lysis buffer and incubated with rhotekin Rho-binding peptide (GST-Rhotekin-RBD) immobilized on agarose to pull down GTP-bound Rho. The expression of activated GTP-Rho and total RhoA was detected using western blotting.
siRNA against RhoA (si-RhoA, sc-29471) and a negative control (si-control, sc-37007) were provided by Santa Cruz Biotechnology (Dallas, Texas, USA). The cells were transfected with the indicated siRNA using a 4D-Nucleofector system (Lonza, Alpharetta, GA, USA) according to the manufacturer’s protocol. After 48 to 72 h, cells were treated with NG or HG and then harvested and analyzed.
Statistical analyses were performed using GraphPad Prism 7.0 software (La Jolla, CA, USA). Data are presented as the mean ± standard error. One-way ANOVA with the Newman–Keuls test for post hoc comparisons was performed to test for differences among multiple groups. Student’s t-test was used for comparisons between two groups. Values of P ≤ 0.05 were considered significant.