Isolation and Treatment of Mouse CASMCs

Isolation of mouse CASMCs was described previously [34 (link)]. Briefly, CASMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Gaithersburg, MD, USA), supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco Gaithersburg, MD, USA) in humidified 100% air and 5% CO2 mixture at 37 °C. Confluent cells (70%–80%) were treated with or without high phosphate (Pi) (3 mmol/L) [35 (link)] and then incubated with TRMPL1 channel agonist, MLSA-1 (10 μM, Sigma-Aldrich Chemicals, St. Louis, MO, USA) and TRMPL1 channel blocker, verapamil (10 μM, Cayman Chemical, Ann Arbor, MI, USA) incubated for 24 h [31 (link),55 (link)].