Growth medium supplement pack

Human embryonic kidney (HEK) 293 T cells and human lung epithelial A549 cells (both originally from ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS) and 100 U/mL of penicillin-streptomycin (Gibco Life Technologies). hTERT-immortalized human lung fibroblasts (MRC-5/hTERT; a gift from Chris Boutell, University of Glasgow, UK) were cultured in Minimum Essential Medium Eagle (Sigma-Aldrich) supplemented with 10% FBS, 100 U/mL of penicillin–streptomycin, 2 mM GlutaMAX (Thermo Fisher Scientific) and 1% non-essential amino acids (NEAA; Thermo Fisher Scientific). Primary human bronchial epithelial cells (HBEpCs) from a 62-year-old male Hispanic donor (Epithelix; donor 1) and from a 52-year-old female Caucasian donor (PromoCell; donor 2) were cultured in Airway Epithelial Cell Growth Medium (PromoCell) supplemented with the Growth Medium Supplement Pack (PromoCell) and Y-27632 dihydrochloride (10 μM, Selleck Chemicals). All cells were maintained at 37 °C and 5% CO₂. IFN-α2 (NBP2-34971) and IFN-γ (NBP2-34992) were purchased from Novus Biologicals.

Primary human cardiomyocytes (HCM) were cultured in Myocyte Basal Medium (C-22270) supplemented with Growth Medium Supplement Pack (C-39270) purchased at Promocell, DE. HEK-293 cells were cultivated in DMEM/F12 medium (Gibco, UK) supplemented with 10% FCS, 1% Penicillin/Streptomycin mix and 1% glutamine (all Gibco, UK). Cells were cultured at 37 °C, 20% O₂ and 5% CO₂ conditions and were split when they reached approximately 70% confluency. One day before transfection, 2 × 10⁵ HCM were plated on a 60 mm tissue culture plate. siRNA was transfected using the Gene Trans II Transfection Reagent (0202B, MoBiTec) with a final concentration of 60 nM in Opti-MEM I serum reduced medium (31985070, Thermo Scientific) according to manufacturer’s instruction. After 4 h, cells were transferred to full growth medium and harvested after a total of 24 h or 48 h. The following siRNA sequences were used for lipofection Scr: UCUCUCACAACGGGCAUUU[dT][dT], siADAR1: GCUAUUUGCUGUCGUGUGA[dT][dT], siADAR2: GAUCGUGGCUUGCAUUAA [dT][dT]. siRNA was produced and predesigned by Merck, DE.

Inhibition of the YAP/TAZ signaling pathway was obtained by using either siRNAs against YAP and TAZ or the small molecule TEAD inhibitor K-975 (Kaneda et al., 2020 (link)). For siRNA-mediated inhibition of YAP/TAZ, HUVECs were transfected with siRNAs using Lipofectamine^TM RNAiMAX reagent (Thermo Fischer) in Opti-MEM and L-Glutamine (Gibco) according to the manufacturer’s instruction. Endogenous YAP/TAZ expression was knocked down by transfecting cells with 100 nM of siRNA against YAP (Silencer-select siRNA s20366, Ambion) and TAZ (ON-TargetPlus siRNA, Horizon discovery). Control cells were transfected with negative control siRNA (Ambion). Cells were incubated with transfection complexes at 37°C in 5% CO2 for 4 h and media was replaced with complete endothelial growth medium for 20 h. Then, HUVEC were cultured for additional 24 h on either normal (5 mM) or high glucose (25 mM) condition in endothelial cell medium added with Growth Medium Supplement Pack (PromoCell). For the purpose of TEAD inhibition, K-975 was synthesized internally based on Kaneda et al. (2020) (link). K-975 at 200 nM was added to HUVECs cultured on either normal (5 mM) or high glucose (25 mM) for 24 h, and under either static conditions or subjected to fluidic conditions. DMSO (0.01%, v/v) was used as control.