Hbmec

Cronobacter sakazakii strains (strain no. ATCC29544, ATCC12868, ATCC29004, and ATCCBAA-894) and human brain microvascular endothelial cells (HBMEC) were purchased from the American Type Culture Collection (ATCC). Bacterial strains were grown at 37°C in brain heart infusion (BHI) broth (Landbridge, Beijing, China) overnight. HBMEC were grown in DMEM (Dulbecco’s modified eagle medium, Gibco, Grand Island, NY, United States) with 10% FBS (Biological Industries, Kibbutz Beit Haemek, Israel) at 37°C and 5% CO₂ for the indicated period of time in different experiments.

Human brain microvascular endothelial cell line (HBMEC) obtained from American Type Culture Collection was grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA), 2 mM glutamine, 1 mM pyruvate, 100 g/mL penicillin, 100 g/mL streptomycin, 1% essential amino acids, and 1% MEM vitamin solution (Gibco, USA). The meningitis-causing E. coli strain RS218, whose genome contains a T6SS gene cluster, was chosen as the model bacterial strain. In addition, a hcp1-deleted mutant RS218 strain, named RS218 Δhcp1, was included as a negative control. Both RS218 and RS218 Hcp1 mutant were grown in LB broth with appropriate antibiotics. The deletion of hcp1 from RS218 was performed based on a previously described protocol (Datsenko and Wanner, 2000 (link); Zhou et al., 2012 (link)).

High BM BC cell lines (MDA-MB-231BM), low BM BC cell lines (MDA-MB-231, SKBRS, MDA-MB-468, MCF-7), normal BC cell lines (MCF10A), human brain microvascular endothelial cell (HBMEC), and human astrocytoma cell (HAC) were obtained from the American Type Culture Collection (Manassas, VA, USA). With the exception of HBMEC cultured in endothelial cell medium (ECM) (ScienCell Research Laboratories, CA, USA), the other cell lines were maintained in DMEM medium (Corning, NY, USA) containing 10% FBS (FBS, ScienCell, USA) plus 100 U/mL penicillin/streptomycin at 37 °C in a 5% CO2 of humid environment.

The human umbilical vein endothelial cells (HUVEC, CRL-1730), human brain microvascular endothelial cells (HBMEC, CRL-3245), human aorta-vascular smooth muscle cells (HA-VSMC, CRL-1999), and human monocytes THP-1 (TIB-202) cell lines were purchased from the American Type Culture Collection (Manassas VA, USA); The oil red O solution was obtained from Beyotime Biotechnology (Shanghai, China); the shRNA lentivirus vector and the polybrene were obtained from the Cyagen Bioscience Inc., (Suzhou, China); IL-12, IL-6, and TNF-α ELISA kits were provided by Boster Biological Technology Co. Ltd. (Wuhan, China); Tissue Total cholesterol Assay Kit was from Applygen Technologies Inc. Beijing, China); PrimeScript RT reagent Kit, SYBR Premix DimerEraser™ (Perfect Real Time) assay kit, and the primers were purchased from Takara (Dalian, China); the primary antibodies p-TNK1 (D46E7), TNK1 (C44F9), p-STAT1 (58D6), STAT1 (D1K9Y), p-Tyk2 (Y1054), and Tyk2 (9312S) were purchased from CST (Danvers, MA, USA); the primary antibody for β-actin (AP0060) was purchased from Bioworld Technology (Nanjing, China); and the oxLDL was provided by the Peking Union-Biology, Co. Ltd (Beijing, China).

Human umbilical vein endothelial cells (HUVECs), human brain microvascular endothelial cells (HBMECs), and immortalised mouse brain endothelial bEnd.3 cells were obtained from the American Type Culture Collection (ATCC). HUVECs, HBMECs, and bEnd.3 cells were grown in the endothelial cell medium (ECM) (Cat. No. 1001, ScienCell, USA) containing 5% FBS (Cat. No. 0025, ScienCell, USA), 1% endothelial cell growth supplement (ECGS, ScienCell, US), and 1% antibiotic solution (P/S, ScienCell, USA). Following 12-h serum deprivation synchronization, HUVECs and bEnd.3 cells were treated with H₂O₂ and NAC supplement. Cells were plated in complete culture media with 10 μM H₂O₂ for 6 h, then exchanged the medium supplemented with 1 mM NAC for another 6 h.
For TET2 siRNA transfection, TET2 siRNAs were commercially acquired from Hanbio Ltd, Shanghai, China (sequences were list in Table 2). HUVECs and HBMECs were seeded in 6-well plates and transfected with control siRNA or siRNA for TET2 using riboFECT™ CP Transfection Kit (Hanbio Ltd, Shanghai, China) according to the manufacturer’s instructions after 70–80% cell confluence was reached. The final concentration of siRNA solution was 20 μM. MISSION^® siRNA Universal Negative Control (Sigma-Aldrich) was used as control.