Ant nr 2

Huh7.5 (cancerous Human hepatocytes, RRID:CVCL_7927), HEK293T (transformed human embryonic kidney cells RRID:CVCL_0063), A549, NIH-3T3 (spontaneously immortalized NIH Swiss mouse embryonic fibroblasts, RRID:CVCL_0594), MCA-RH 7777 (cancerous buffalo rat hepatocytes, RRID:CVCL_0444), FEA (spontaneously immortalized cat embryonic fibroblasts, RRID:CVCL_UG17), CRFK (spontaneously immortalized cat kidney epithelial cells, RRID:CVCL_2426), and Vero (spontaneously immortalized green monkey kidney epithelium, RRID:CVCL_0059) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco #41965–039) supplemented with 10% heat-inactivated fetal calf serum (Sigma-Aldrich #F75424), and 100 μg/mL of normocin (InvivoGen #ant-nr-2) and grown in cell culture flasks at 37°C in a humidified incubator containing 5% CO2. I/1Ki (boa constrictor kidney cells)[83 (link)] and I/1Ki-Δ (referred to as I/1Ki-SDeV in this paper) [13 (link)] were maintained in Minimal Essential Medium Eagle (MEM; Gibco #31095–029) supplemented with 20% heat-inactivated fetal calf serum (Sigma-Aldrich #F75424), and 100 μg/mL of normocin (InvivoGen #ant-nr-2), and grown in cell culture flasks at 30°C in a humidified incubator containing 5% CO2. All cell lines used in the study were mycoplasma-free and were checked regularly for mycoplasma contamination.

Early and mid-gestation human fetal brain endothelial cells (hfBECs) were isolated as previously described [9 (link)]. In brief, after isolation from fetal brains, cells were plated on type I rat tail collagen- (50 μg/mL; 5056, Advanced BioMatrix, San Diego, CA, USA) coated tissue culture flasks (353136, ThermoFisher scientific, Mississauga, ON, CA) and grown in a 37 °C/5% CO₂-incubator in EndoGROTM-MV Complete Culture Media Kit®, (SCME004, Millipore, Blvd, ON, Canada), supplemented with recombinant human epidermal growth factor (5 ng/mL), L-Glutamine (10 mM), hydrocortisone hemisuccinate (1.0 µg/mL), heparin sulfate (0.75 U/mL), ascorbic acid (50 µg/mL), 20% FBS, penicillin (100 IU/mL), streptomycin (100 IU/mL) (15,140–122, Life Technologies, Carlsbad, California, USA), 1% normocin antibiotic (ant-nr-2, Invivogen, San Diego, CA, USA) at 20% O₂ (5% CO₂, 37 °C). hfBECs were used at passage 4 in all subsequent experiments, and subjected to PAMP treatments as described below.

Human cancer cell lines used at MD Anderson were obtained as follows: EOL1, MONOMAC1, NB4, OCIAML3 (DSMZ, #ACC-386 #ACC-252 #ACC-207 #ACC-582); MOLM13 and NOMO1 (Fisher, #NC0442994 #NC1515509); MV411 (ATCC #CRL-9591). Identities were confirmed upon receipt and prior to experiments by STR typing (MDACC Characterized Cell Line Core). The absence of mycoplasma was confirmed monthly (Invivogen #rep-pt1). All cell lines were grown at 37 °C in 5% CO₂ in low attachment flasks (Greiner) and maintained at less than 1 M cells ml⁻¹. All but one line were cultured in RPMI-1640 with 25 mM HEPES ((Sigma #R5886) supplemented with 10% FBS (Sigma # F0926), 2 mM Glutamax (Gibco #35050061), 1 mM sodium pyruvate (Gibco #11360070), 10,000 units ml⁻¹ penicillin (Gibco #15140122), 10 mg ml⁻¹ streptomycin (Gibco #15140122), and 100 µg ml⁻¹ Normocin (Invivogen #ANTNR2). Complete medium was additionally supplemented with 0.1 mM nonessential amino acids (Gibco #11140050) for MONOMAC1.