Vascular smooth muscle cell growth kit

Cardiomyocytes derived from human induced pluripotent stem cells (iPSCs, ATCC-ACS-1021) were cultured as instructed. For cardiomyocyte differentiation in vitro, a STEMdiff ventricular cardiomyocyte differentiation kit (STEMCELL Technologies, cat# 05010) was used according to the manufacturer’s instructions. Cardiomyocytes were cultured for 15 days in maintenance medium (STEMCELL Technologies, cat# 05020) to promote maturation. To induce hypertrophy, cardiomyocytes were treated with 10 nM of endothelin 1 (ET-1, Sigma-Aldrich, cat#E7764) for 24 h. Human pulmonary artery smooth muscle cells (PASMCs) were purchased (ATCC, cat# PCS-100-023) and cultured in vascular cell basal medium (ATCC, cat# PCS-100-030) supplemented with a vascular smooth muscle cell growth kit (ATCC, cat# PCS-100-042) in 5% CO₂ at 37^°C. Cells from passages 4–7 were used for experiments. Sotagliflozin (Selleckchem, cat# S8103) was used as described for each experiment.

WT and their littermate mgR mice at 7 days old were anesthetized and underwent thoracotomy. Mouse thoracic aortas were isolated and minced. SMC isolation was described previously [22 (link)]. The cells were grown to confluence and passed after trypsinization with 0.25% trypsin. Mouse SMC were maintained using the Vascular Smooth Muscle Cell Growth Kit (ATCC). Experiments were performed on cells from passage 2–4.

Human primary aortic smooth muscle cells were purchased from American Type Medium (ATCC PCS‐100‐030) with Vascular Smooth Muscle Cell Growth Kit (ATCC PCS‐100‐04). Lipofectamine 3000 (Invitrogen) and SuperFect Transfection Reagents (Qiagen) were used for plasmid and siRNA transfection.

Human primary uterine myometrial cells (HutSMCs) were obtained from ATCC. Cells were maintained in Vascular Cell Basal Medium (PCS-100-030, ATCC) supplemented with Vascular Smooth Muscle Cell Growth Kit (PCS-100-042, ATCC) and cultured in an incubator with 5% CO₂ at 37°C. All the experiments were performed on myometrial cells at the third to the sixth passage. The K-562 (human myelogenous leukemia cell line) cells were grown in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS.
N-Formyl-Met-Leu-Phe (fMLF, agonist of FPR1, MCE, United States) and Boc-MLF TFA (tBOC, antagonist of FPR1, MCE, United States) were dissolved in dimethyl sulfoxide (DMSO, Sigma, United States) to a final concentration of less than 0.1%. After reaching 80% confluence, HutSMCs were serum-starved for 12 h followed by treatment with DMSO, fMLF (10 μM), tBOC (5 μM) or a combination of fMLF and tBOC for 24 h. The concentrations of these two reagents are non-toxic dose to cells, and its election was based on preliminary experiments (Supplementary Figure 1).

hiPSC-derived VSMCs were purchased from ImStem Biotechology (Farmington, CT, USA). hiPSC-derived VSMCs were chosen to ensure an adequate amount of the same cells for all experiments. Primary rat VSMCs were prepared by using the enzymatic digestion of thoracic arteries from 3-week-old Sprague–Dawley rats. Transfection reagents were obtained from ThermoFisher Scientific (Waltham, MA, USA), except for siRNA targeting rat ENPP1, which was obtained from Sigma-Aldrich (St. Louis, MO, USA). Cells were seeded in a collagen type I-coated 60 mm dish or a regular polystyrene dish at a density of 3500 cells/0.32 cm² in Smooth Muscle Growth Medium-2 (Lonza, Allendale, NJ, USA) or Vascular Cell Basal Medium (ATCC, Manassas, VA, USA), which is contained in the Vascular Smooth Muscle Cell Growth Kit (ATCC). After overnight culture, either siRNA targeting human ENPP1 s10264 (Cat 4390824) and control siRNA (Cat 4390846) or siRNA targeting rat ENPP1 (SASI Rn01_00111206) and control siRNA (Cat 4390847) were transfected into hiPSC-derived VSMCs or rat VSMCs, respectively, using Lipofectamine RNAiMAX overnight, according to the manufacturer’s instructions.