M 199 growth medium

Bovine aortic endothelial cells (BAECs) were used between passages 4–6. Upon 80% confluence, cells were placed in M-199 growth medium (Invitrogen) containing normal glucose (NG, 5.5 mmol/L) or high glucose (HG, 25 mmol/L), 0.2% fetal bovine serum, 50 μmol/L _L-arginine, 100 U/ml penicillin, 100 μg/ml streptomycin and _L-glutamine for periods of 15, 30, 45, and 60 min or 24 and 48 hrs. Another group of cultures was pretreated with netrin-1 (100 ng/ml) for 1 hr before the addition of NG, HG or mannitol as osmotic control medium (5.5 mmol/L D-Glucose + 19.5 mmol/L mannitol) for periods of 1 to 48 hrs. At the end of the treatment periods, cells were harvested for enzymatic assay and western blot analysis.

Breast cancer cell lines MDA-MB231 (MDA-231), MCF-7, and HUVEC were purchased from American Type Culture Collection (ATCC, USA). GFP⁺ECs (ECs) were developed as described previously [21 (link)]. Human recombinant Jagged1 and TGFβ1 were obtained from R&D Systems and PeproTech, respectively. Υ-secretase inhibitors (GSI) and SB-431542 were purchased from Sigma (USA). Breast cancer cells (BCCs) were grown in DMEM/High glucose (HyClone, USA) supplemented with 10% FBS, L-glutamine, non-essential amino acids (NEAA), and penicillin/streptomycin in a humidified incubator with 5% CO₂. ECs were grown in M199 growth medium (Gibco, USA) supplemented with 20% FBS, 20 ng/ml β-Endothelial Cell Growth Factor (βECG), 20 units/ml heparin and penicillin/streptomycin. The co-cultures were prepared by mixing one part BCCs with 10 parts GFP⁺ECs (1:10 ratio) and cells were grown in 1:1 ratio of DMEM/High and M199 media in the absence of serum and growth factors (complete starvation). Co-cultivation of BCCs and ECs was performed over 3–5 days under adherent condition.

Human AVICs were isolated and cultured as previously reported [23 ]. Briefly, after the collagenase digestion of aortic valve leaflets, cells were collected by centrifugation. We cultured AVICs in M199 growth medium (Gibco, C11150500BT, USA), which contains 100 units of penicillin G, 100 μg of streptomycin, and 10% foetal bovine serum. Following passage 3, cells reached 80%-90% confluence and were then used in subsequent experiments. For osteogenic differentiation, cells were cultured with osteogenic medium (OM) (growth medium supplemented with 10.0 mmol/L β-glycerophosphate, 10.0 nmol/L dexamethasone, 4.0 μg/ml cholecalciferol and 8.0 mmol/L CaCl₂) [24 (link)]. For some experiments, cells were transfected with a control vector (pCMV3-control, SinoBiological, Beijing, CN), PTPN22 expression vector (pCMV3-PTPN22, SinoBiological, Beijing, CN), or PTPN22-specific small interfering RNA (siRNA, OBIO Biotechnology, Shanghai, CN) by Lipofectamine 3000 (ThermoFisher, L3000015, USA) according to the manufacturer’s protocol. Scramble RNA was employed as negative control. Human AVICs were then treated with S18 as indicated.

All-trans-Retinal (Sigma) was first dissolved in filtered DMSO (Sigma) to a stock concentration of 100 mM, and further diluted in M199 growth medium (GIBCO) with 10% or 2% FBS to final concentration of 1 to 10 μM. Prior literature shows that concentrations of <0.02% DMSO do not have undesirable effects on neurons nor cardiomyocytes42 (link)43 (link). The growth media supplemented with ATR was filtered again before use. CMs were grown in ATR-containing medium with replenishment as described, up to the time of viability assay and experimentation. The experimental groups included both control and Ad-ChR2(H134R)-eYFP-infected samples, treated with the selected ATR concentration (0, 0.1, 0.5, 1, 2, 4, 8, and 10 μM), i.e. 16 groups total.

Human cerebral microvascular endothelial cells (hCMVECs) are a cell line, immortalised by SV40 large T antigen, and purchased from Applied Biological Materials Inc. (ABM, cat# T0259, Richmond, BC, Canada) The cells were cultured in Nunc T75 flasks (cat# 156499) and provided with M199 growth medium (cat# 11150-067, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS, 1 μg/mL hydrocortisone (cat# H0888, Sigma-Aldrich, St. Louis, MO, USA), 3 ng/mL hFGF (cat# PTAF10018B50, Peprotech, Rocky Hill, NJ, USA), 1 ng/mL hEGF (cat# PTAF10015100, Peprotech, Rocky Hill, NJ, USA), 10 μg/mL heparin (cat# H-3393, Sigma-Aldrich, St. Louis, MO, USA), 2 mM GlutaMAX (cat# 305050-061, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 80 μM dibutyryl-cAMP (cat# D0627, Sigma-Aldrich, St. Louis, MO, USA), later referred to as complete M199 medium. For both cell maintenance and experiments, the flasks/plates were coated with 1 μg/cm² collagen I (cat# A1048301, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) dissolved in 0.02 M acetic acid for 1 h and washed three times with sterile MilliQ water, prior to cell plating [1 (link)].