Dulbecco s modified eagle s medium f 12

Target sites for genome editing were chosen using the web tool CRISPRdirect (https://crispr.dbcls.jp/) to minimize the possibility of off-target cleavage (83 (link)). The information about the chosen target sequences is summarized in Fig. S5. 293T cells were seeded into 24-well plates in Dulbecco's modified Eagle's medium/F-12 (FUJIFILM Wako) containing 10% fetal bovine serum (FBS). After a day, cells were transfected with the SpCas9-2A-Puro expression plasmid and sgRNA expression plasmid using HilyMax (DOJINDO) according to the manufacturer’s instructions. Six to eight hours after transfection, the medium was replaced with a fresh medium. Twenty four hours after transfection, puromycin was supplied at 5 μg/ml. Twenty four hours after the addition of puromycin, live cells were trypsinized, counted, and seeded again on a 10-cm culture dish at 100 to 300 cells per dish. After 15 days, clonal colonies on the culture dish were scratched and transferred to 24-well plates. Genotyping was performed by PCR for each clone to identify the cells carrying biallelic Gα gene KO. The GNAQ/GNA11 double–KO cell was generated by sequentially repeating the above process twice. Thereafter, the GNAQ/GNA11/GNA14/GNA15 quadruple KO cell was produced by one step of the same procedure, involving sgRNA expression plasmids targeting both GNA14 and GNA15.

Antibodies were obtained from the following suppliers: anti-Mas receptor (AAR-013) (Alomone Laboratories, Ltd., Jerusalem, Israel); anti-endothelial nitric oxide synthase (eNOS) (BD Biosciences, Mississauga, ON, Canada); anti-aryl hydrocarbon receptor (AhR) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-α-tubulin (Calbiochem, La Jolla, CA, USA); anti-signal transducer and activator of transcription 3 (Stat3), anti-phosphorylated eNOS (Ser1177) (peNOS), anti-transforming growth factor-β1 (TGF-β1), anti-rabbit IgG horseradish peroxidase-linked antibody and anti-mouse IgG horseradish peroxidase-linked antibody (Cell Signaling Technology, Beverly, MA, USA). IS was obtained from Alfa Aesar (Morecambe, Lancs., UK). Ang-(1–7) was purchased from Bachem California (Torrance, CA, USA). N-acetylcysteine (NAC), an antioxidant, was from Calbiochem (La Jolla, CA, USA). Dulbecco’s modified Eagle’s medium/F12 was purchased from Wako (Osaka, Japan). Trypsin-EDTA, fetal bovine serum, and insulin transferrin-selenium were purchased from Gibco (Grand Island, NY, USA). Penicillin and streptomycin were purchased from Nacalai Tesque Inc. (Kyoto, Japan).