Hcmec

HCMECs were purchased from Sciencell Research Laboratories and maintained in EC medium (Sciencell, California, U.S.A.) as described recently [27 (link)]. A density of 10000 cells/cm² of passages 2–6 were seeded in a six-well plate. Cells were divided into five groups including – (i) control group: HCMECs were cultured under normoxia conditions; (ii) hypoxia group: HCMECs were cultured under hypoxia conditions (1% O₂) for 3 days; (iii) hypoxia + rapamycin group: HCMECs were pretreated with rapamycin (100 nM, Sigma–Aldrich, St. Louis, MO, U.S.A.) for 2 h and cultured under hypoxia conditions (1% O₂) for 3 days; (iv) hypoxia + 3-methyladenine (3-MA) group: HCMECs were pretreated with 3-MA (5 mM, Sigma–Aldrich, St. Louis, MO, U.S.A.) for 2 h and cultured under hypoxia conditions (1% O₂) for 3 days; and (v) hypoxia + chloroquine (CQ) group: HCMECs were pretreated with CQ (20 μM, Sigma–Aldrich, St. Louis, MO, U.S.A.) for 2 h and cultured under hypoxia conditions (1% O₂) for 3 days.

Human brain microvascular endothelial cells (hCMEC/D3 or hCMEC) from Millipore Sigma (Burlington, MA) (passage 7 to 15 after purchase) were cultured in EBM-2 MV endothelial cell growth basal Medium (Lonza, Basel, Switzerland), supplemented with 100 U/ml penicillin-Streptomycin (Gibco, ThermoFisher, Waltham, MA). Human breast carcinoma cells (MDA-MB-231 or MB231) from ATCC (Manassas, VA) (passage 8 to 18 after purchase) were cultured in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin sulfate, all from Sigma-Aldrich. Both cells were incubated in the humidified atmosphere with 5% CO₂ at 37 °C.²⁴ (link)

Human cerebral microvascular endothelial cells (hCMEC/D3 or hCMEC) from Millipore Sigma (Burlington, MA, USA) (passage 7 to 20 after purchase) were cultured using EBM^TM-2 Basal Medium (Lonza, Basel, Switzerland), supplemented with EGM^TM-2 MV Microvascular Endothelial Cell Growth Medium SingleQuots^TM kit (Lonza) [49 (link)]. Human breast carcinoma cells (MDA-MB-231 or MB231) from ATCC (Manassas, VA, USA) (passage 10 to 18 after purchase) were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12), 2 mM L-glutamine, 100 U/mL penicillin, and 1 mg/mL streptomycin, all from Sigma-Aldrich (St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA, USA) [26 (link),27 (link),28 (link)]. Human nontumorigenic breast epithelial cell line MCF-10A cells (ATCC) were cultured in MEGM bullet kit (Lonza) supplemented with 100 ng/mL cholera toxin (Sigma-Aldrich) as described in [26 (link)]. All cells were cultured in the incubator with 5% CO₂ at 37 °C.