Cholesterol c3045

Human umbilical vein endothelial cells (HUVECs) were obtained from Science Cell Research Laboratories (Carlsbad, CA, USA) and cultured in Endothelial Cell Medium (ECM, Science Cell Research Laboratories, Carlsbad, CA, USA) supplemented with 1% endothelial cell growth factors, 5% FBS, and 1% penicillin/streptomycin at 37°C in a 5% CO₂ humidified atmosphere. Cholesterol (C3045) and total ROS scavenger N-acetyl-L-cysteine (NAC, A7250) were purchased from Sigma-Aldrich; colchicine (HY-16569) was obtained from MCE. For experiments involving pharmacological reagents, endothelial cells were pretreated with colchicine (0-100 nM) or NAC (5 mM) for 2 h and subsequently followed by cholesterol crystals (0.5 mg/ml) for 10-24 h in the presence of these reagents.

Cholesterol (# C-3045) was purchased from Sigma (St. Louis, MO, USA). d-tagatose (FW 180.16, cat #T1501), d-fructose (FW180.16, cat # F0060), and d-psicose (FW 180.16, cat # P1699) were purchased from Tokyo Chemical Industry (Tokyo, Japan).

MβCD (C4555) and cholesterol (C3045) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Filipin III, a cholesterol probe, was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). The primary antibodies used for immunofluorescence assay were as follows: anti-CAV1 (1:400; D46G3 rabbit IgG mAb; Cell Signaling Technology, Danvers, MA, USA), anti-PTEN (1:100; clone 17A, mouse IgM mAb, Lab Vision, South San Francisco, CA, USA), and anti-integrin β1 (ITGB1) (1:100; P5D2, mouse IgG mAb, Chemicon International, Temecula, CA, USA). Alexa Fluor^TM 488-conjugated goat antibodies against rabbit IgG (1:500), Alexa Fluor^TM 488-conjugated goat antibodies against mouse IgG (1:500), Alexa Fluor^TM 568-conjugated goat antibodies against rabbit IgG (1:500), and Alexa Fluor^TM 555-conjugated goat antibodies against mouse IgM (heavy chain) (1:500) were used as the secondary antibodies and were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Rhodamine-phalloidin conjugate (1:500; Thermo Fisher Scientific, Waltham, MA, USA) was used for F-actin staining, and Hoechst 33342 (1:500; Dojindo Laboratories Co., Ltd., Kumamoto, Japan) was used for nuclear staining.