Human angiotensin 2

HEK 293 T cells were seeded into T75 flasks at 90,000 cells/cm² and incubated at 37 °C and 5% CO₂ overnight. The following day, cells were transiently transfected for 48 hours in total; for the last 24 hours, cells were plated at 10,000 cells/well into sterile, white, 384-well plates (Nunc/Thermo Fisher Scientific) containing 5–10 mg/ml of EMR2 antibody or 25 μM AngII (human Angiotensin II, Sigma-Aldrich) in phenol-red free OptiMEM1 medium (Gibco/Thermo Fisher). Luminescence was measured using the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA) as per manufacturer’s instructions. The Luminescent signal was read on a EnVision imager (Perkin-Elmer).

Human angiotensin II was purchased from Sigma-Aldrich (St. Louis, MO, USA). The lignan content in Eucommia lignans was determined to be 71% by spectrophotometry on a Beckman Coulter DU 640 spectrophotometer (Beckman Coulter, Inc., USA) at 277 nm; the lignans were extracted from Eucommia ulmoides Oliv. bark in our own laboratory using a previously described method [2 (link)].
The primers for P21, P27, Bax, Bcl-2, and AR were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against P21, P27, Bax, Bcl-2, and AR for western blotting were supplied by Abcam (Cambridge, England) and Santa Cruz (CA, USA). The Cell Titer 96 Aqueous One Solution Proliferation Assay for the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method was purchased from Promega (Madison, WI, USA). The RT-qPCR kits with Platinum SYBR Green qPCR Super Mix-UDG and flow cytometry were purchased from Invitrogen (Carlsbad, CA, USA). Epalrestat was obtained commercially from Yuancheng Pharmaceutical Co., Ltd. (Hubei, China). The MTT One-Step Cell Proliferation Test Kit and Annexin V-FITC Apoptosis Assay Kit were purchased from the Promega Corporation and Bibo Biological Technology Co., Ltd. (Nanjing, China), respectively.