Endothelial cell growth medium 2 bulletkit

Primary HUVECs (Lonza Japan, Tokyo, Japan) were obtained and cultured using an endothelial cell growth medium-2 bullet kit (Lonza, Tokyo, Japan) containing growth factors, supplements and 5% fetal bovine serum (FBS). The culture medium was changed every 24 h. When HUVECs were 70–80% confluent, the cells were trypsinized, resuspended in the culture medium and seeded into 96-, 24-, or 6-well microplates for each assay and fluorescence microscopy. HUVECs in the third passage were used for experiments.
When HUVECs were 90% confluent, the medium was exchanged with endothelial cell basal medium-2 (Lonza, Tokyo, Japan) and incubated with 7.5% serum from KD patients or patients with bacterial infections. After culturing of HUVECs for 24 h, IG (Venoglobulin-IH^TM, Japan Blood Products Organization, Tokyo, Japan) was added at a concentration of 20 mg/ml. For those cells receiving combination therapy, PSL (Predonine, Shionogi, Osaka, Japan) was added at a concentration of 10⁻⁶ M per well; subsequently, wells were cultured in a 5% CO₂ incubator for 24 h at 37 °C. All prepared culture media were maintained at a final pH of 7.2 to 7.4. The decision to use IG at a concentration of 20 mg/ml and PSL at concentration of 10⁻⁶ M was based on the concentrations used in a previous report48 (link). All experiments were repeated at least three times.

Pig aortas were incubated with 0.005% collagenase type IV (sigma, USA), and the isolated cells were washed with DPBS(Gibco). The cells were then culture with endothelial cell growth medium-2 bullet kit (Lonza, Switzerland). Pig corneal endothelial cells were isolated and cultured, as described in our previous study^{80 (link)}. Pig kidney tissues were minced into 1–2-mm sections, and digested with 3% collagenase type IV. The digested tissues were passed through a 100-m cell strainer (BD). The isolated cells were washed with DPBS (Gibco), and cultured in DMEM (Welgene) containing 15% FBS (Hyclone), 1% non-essential amino acid, 0.1 mM β-mercaptoethanol, and 1% antibiotic–antimycotic (Gibco).

H9c2 cells were cultured in Dulbecco’s modified eagle medium (Gibico, Waltham, CA) supplemented with 10% fetal bovine serum (Gibico, Waltham, CA) and 1% of penicillin/streptomycin at 37°C in an incubator with 95% humid air and 5% CO₂ (Wang M. et al., 2020 (link)). Human umbilical vein endothelial cells (HUVECs) were maintained in endothelial cell growth medium-2 bullet kit (Lonza, Basel, BS, CH) at 37°C in an incubator with 95% humid air and 5% CO₂ (Chen et al., 2020 (link)). To simulate myocardial ischemia, H9c2 cells and HUVECs were managed with hypoxia. Specifically, they were placed in a hypoxic incubator containing 94% N₂, 5% CO₂, and 1% O₂ for 24 h. Meanwhile, the control group was maintained in a normal atmosphere of 95% air and 5% CO₂ at 37°C (Zhu et al., 2021 (link)). Afterward, H9c2 cells and HUVECs were cultured in consistent 1% O₂, 2% O₂, and 5% O₂ hypoxic condition for 24 h to explore the impact of different concentrations of oxygen on m⁶A methylation level. These cells were also cultured with 1% oxygen concentration for 12, 24, and 48 h to explore the impact of different hypoxic duration on m⁶A methylation level. H9c2 cells and HUVECs were then treated with 25 μM Mettl3 inhibitor (STM2457) for 24 h to explore the role of Mettl3 on m⁶A methylation level (Yankova et al., 2021 (link)).

Human umbilical vein endothelial cells (HUVECs, Lonza) were cultured with Endothelial Cell Growth Medium-2 BulletKit (Lonza). For evaluation of NO production, HUVECs were incubated with 500 μM palmitic acid and 10 μg/ml BP or 10 μg/ml AC in FBS-free medium for 6 hr. Then the medium was exchanged for FBS-free medium with 5μM DAR-4M and incubation was continued for 30 min. Subsequently, 100 nM insulin was added and the HUVECs were incubated for a further 30 min, after which the cells were fixed with 4% PFA and observed. For evaluation of ROS production, HUVECs were pre-incubated with 10 μg/ml BP or 10 μg/ml AC for 2 hr, after which 500 μM palmitic acid was added and the cells were incubated for 15 min. Subsequently, the HUVECs were fixed with 4% PFA and incubated with DHE for 30 min at 37°C. Stock solution of palmitic acid (5 mM) was prepared by conjugation with 10% BSA.

Uninfected and A. phagocytophilum (NCH-1 strain) infected human promyelocytic HL-60 cells [ATCC, CCL-240; American Type Culture Collection (ATCC)] and Macaca mulatta RF/6A choroidal endothelial cells (CRL-1780; ATCC) were cultured as described in (71 (link)). HL-60 and RF/6A cells pretreated with 300 nM and 400 nM of NVP231 [Cayman Chemical, (catalog #13858)], respectively, 50 µM of SP006125 [Sigma Millipore (372770)], 3 µM of Gö6976 (Sigma Aldrich, 372770), or 0.001% of DMSO for 1 h were incubated with A. phagocytophilum as described in reference (70 (link)). HUVECs (Lonza, CC-2519) were cultivated in Endothelial Cell Growth Medium-2 Bullet Kit (Lonza) in a humidified incubator at 37°C with 5% atmospheric CO₂.

Human dermal microvascular endothelial cells (HDMECs) (Lonza, Walkersville, MD, USA) were cultured on collagen-coated tissue culture plates in Endothelial Basal Medium-2 supplemented with the Endothelial Cell Growth Medium-2 Bullet Kit (Lonza). Shortly after seeded, HDMECs were transfected with FLI1 siRNA or non-silencing scrambled RNA (SCR) (10 nM, both purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA) mixed with HiPerFect Transfection Reagent (Qiagen, Valencia, CA, USA) for 48 h. Some cells were stimulated with recombinant human IL-17A, IL-10, and TGF-β1 (all from Peprotech, Rocky Hill, NJ, USA). Cells were then collected using TRIzol Reagent (Thermo Fisher Scientific) for RNA isolation.