Crl 2922

TEM assay was performed using the CytoSelect Tumor TEM assay kit (CBA-216, Cell Biolabs, Inc. San Diego, CA, USA). Briefly, HUVEC cells (25,000–50,000) (CRL-2922, ATCC, Manassas, VA, USA) were allowed up to 72 hours to form a monolayer on the upper surface of the membrane inside the insert and then activated by overnight serum starvation or TNFα treatment. The 10A-iK8 or 231-K8ikd cells (50,000) were incubated with the Cytotracker dye for 1 hour prior to further incubation in the insert on top of the HUVEC monolayer in the presence or absence of doxycycline. Depending upon specific experimental need, AMD3100 (35 ng/ml) was included to inhibit CXCR4 prior to and during the 22-hour assay time. The cells that migrated through the HUVEC monolayer were lysed and quantified using a Fluorescence multi-plate reader (PolarStar Omega, BMG LabTech, Cary, NC, USA).

To study S. aureus interaction with endothelial cells we used the EA.hy926 cell line (ATCC® CRL-2922™), originally derived from human umbilical vein. EA.hy926 cells were grown in Dulbecco's modified Eagle high glucose medium (DMEM, Dominique Dutscher™) supplemented with 10% fetal bovine serum (FBS, Eurobio™). Culture media contained 1% penicillin/streptomycin and 1% amphotericin B. Antibiotics were removed from culture medium prior to infection.
The human monocytic cell line THP-1 (ATCC® TIB-202™) was grown in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 5% FBS. Two days before infection, phorbol myristate acetate 200 ng mL⁻¹ (PMA) was added to the culture medium to induce monocytes differentiation into macrophages.
All cells were incubated in a humidified 5% CO2 atmosphere at 37°C.

EA.hy926 and HeLa cells (obtained from ATCC^® CRL-2922™, somatic cell hybrid established by fusing primary HUVEC with a thioguanine-resistant clone of A549; ATCC^® CCL-2, cervical cancer cells; passage No. 15-20; VA, USA) were cultured in Dulbecco’s Modified Eagle Medium; 4.5 g/L Glucose (DMEM; Thermo Fisher Scientific, MA, USA) containing 10% FCS (0.1% FCS under starvation) and 100 U/mL Penicillin and 100 µg/mL Streptomycin in a 37 °C humidified incubator in the presence of 10% CO₂. EA.hy926 reporter cells (NRF2-ARE-mCherry fluorescence based, see Figure 4a) were generated by transfection of plasmid (CS-13227-LvGN01; GeneCopoeia, MD, USA) by FuGene HD Transfection reagent (Promega, WI, USA). Transfection efficiency was improved by transfection of linearized and shortened plasmid (NsbI-AjuI-digest; NEB, MA, USA). Positive cells were selected by puromycin treatment (0.25 µg/mL) and sorted by flow cytometry (BD FACS Aria2; NJ, USA; more than 4-fold over background) after stimulation with 20 ng/mL Phorbol 12-myristate 13-acetate (Sigma-Aldrich, MO, USA). On 6-well dishes, 3 × 10⁵ cells per well were seeded and, where indicated, cells were incubated with different NRF2-inducers (5 µM, 10 µM sulforaphane; LKT Laboratories, MN, USA; and 4 µM, 8 µM falcarinol; Cayman chemical, MI, USA) or inhibitors (10 nM, 30 nM, 100 nM, 300 nM brusatol; Carbosynth, UK).

The human vascular endothelial cell line CRL-2922 (ATCC, Manassas, VA, USA) was cultured in DMEM with 4500 mg/L glucose and supplemented with 15% fetal bovine serum in a 5% carbon dioxide cell incubator at 37°C. For the co-immunoprecipitation and western blot analyses, the cells were seeded in 25-mL culture flasks. For the monolayer cell permeability assay, the cells were seeded on polycarbonate membranes in 24-well Transwell chambers. For immunocytochemistry, the cells were seeded on cover slips. The experiments were performed after the cells reached confluence.

The immortalised cell line EA.hy926 (CRL-2922; ATCC, Manassas, VA, USA) was used in this study. It is a somatic cell hybrid type with endothelial cell morphology, displaying Weibel–Palade bodies that are specific to vascular endothelium, as previously described [15 (link)]. This cell line was derived by fusing primary human umbilical vein cells with a lung carcinoma A549 cell line. EA.hy926 cells (deposited by CS. Edgell) were purchased from ATCC (CRL-2922™).
The EA.hy926 cells were seeded into T25 flasks (Sarstedt, Nümbrecht, Germany) or slide flasks (Nunc™ Lab-Tek™ SlideFlask; Thermo Fisher Scientific, Waltham, MA, USA) for immunofluorescence staining and cultured using DMEM medium (D4947, Merck), supplemented with 10% FBS (F7524, Merck, Damstadt, Germany) and 1% penicillin/streptomycin solution (P4333, Merck, Damstadt, Germany), corresponding to low glucose (LG) medium. Half of the flasks were grown in hyperglycaemic conditions with high glucose (HG) medium, which was pre-made by supplementing regular 5 mM DMEM medium with D-(+)-Glucose solution (G8769, Merck, Damstadt, Germany). The cells were cultured either in 1g or s-µg conditions with either LG or HG medium for 2 weeks. The medium was changed once after seven days. The experimental length was selected to recreate real µg experiments in space, where good cell viability was demonstrated for at least 14 days [16 (link)].

The murine 3T3-L1 preadipocyte cell line was purchased from the Bioresource Collection and Research Center (BCRC #60159; Hsinchu, Taiwan). This cell line is widely used as a model for studying adipocyte differentiation and biology [21 (link)]. Cells were grown and maintained in DMEM supplemented with 10% FBS at 37 °C in 5% CO₂. To induce adipocyte differentiation, preadipocytes were cultivated in a differentiating medium containing insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine for 2 day and then maintained in an insulin-containing medium for another 6 day to obtain round-shaped mature adipocytes. The medium was then replaced by 1% DMEM in the presence of α-NF (1–5 μM) for 16 h, and the adipocytes and culture medium were collected for analysis.
To determine tube formation, a human endothelium-derived EA hy 926 cell line with vascular EC characteristics was used. This cell line was kindly provided by Dr. Cora-Jean Edgell (University of North Carolina, Chapel Hill, NC, USA), who established and characterized the cells [22 (link),23 (link)], and it is now commercially available at ATCC (ATCC^® CRL-2922). The cells were grown as monolayers and maintained in DMEM supplemented with 10% FBS at 37 °C in 95% air and 5% CO₂.