Complete ebm 2 medium

The MEG-01 (human megakaryoblastic cell line, ATCC) and THP-01 (human monocytic cell line from the collection of Moscow State University https://human.depo.msu.ru, accessed on 3 September 2018) cell lines were cultured in RPMI-1640 complete growth medium (Thermo Fisher, Carlsbad, CA, USA) containing an antibiotic-antimycotic (HyClone, Marlborough, MA, USA) and 10% fetal bovine serum (FBS, HyClone). Primary human umbilical vein endothelial cells (HUVEC, https://human.depo.msu.ru, accessed on 3 September 2018) were cultured in EBM-2 complete medium with additives (Lonza, Basel, Switzerland), an antibiotic-antimycotic (HyClone), and 2% FBS (HyClone). Chinese hamster ovary (CHO) cells were cultured in Dulbecco’s modified Eagle’s medium-F12 (HyClone or PanEco) with an antibiotic-antimycotic solution (HyClone) and 10% FBS (HyClone). All cells were passaged by treatment with the Versene solution (PanEco, Moscow, Russia) and HyQTase protease mixture (HyClone). All cell lines were cultured in an incubator at 37 °C and 5% CO₂.

Briefly, adLECs p2 or jdLECs p3 were seeded and expanded for 4 days in a 10 cm culture plate in EBM2 complete medium (Lonza). On the day of the transduction with lentivirus particles, containing one of four different shRNAs or the scrambled control, LECs cells were seeded into 5x 4 wells of a 6-well plate at a concentration of 50 000 cells/ well. (Four wells for each of the ACKR3 shRNAs (A-D) and the scramble control, packaged into Lentivirus particles). After 5h, LECs were infected with ACKR3- shRNA Lentivirus particles (A-D) using a calculated MOI of 10. Four days after transduction, cells were harvested and seeded into a T75 cell culture flask for expansion. Eight days after the Lentivirus infection, LECs were harvested and GFP positive cells were sorted on a FACSAria II (BD Biosciences) cell sorter with a 100μm nozzle, using FACSDiva software (version 6.1.3). Afterwards, cells were expanded and cryopreserved at p6, as a stock for subsequent experiments. Experiments were performed at p7 and p8. The following commercially available shRNA constructs in pGFP-C-shLenti-particles (Origene) were used for shRNA mediated silencing of ACKR3:

B2B cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). B2B cells were transformed into As-T cells by exposure to 1 μM sodium arsenic for 6 months as we previously described, which act like lung cancer cells and can form tumors in nude mice [29 (link)]. Passage-matched B2B cells were used as parental control cells. B2B and As-T cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 50 U/ml Penicillin-Streptomycin. Human umbilical vein endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HMVEC) were obtained from Lonza (Lonza, Hayward, CA, USA) and cultured in endothelial basal medium-2 (EBM-2) complete medium (Lonza). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in the air.

As a general in vitro model of the human BBB, we used hCMEC/D3 cells [15 (link)]. hCMEC/D3 were cultured in tissue culture flasks previously coated with 1 μg/ml rat tail collagen type I solution, in EBM-2 complete medium (Lonza). Cultures were maintained at 37 °C in 5% CO₂, replaced with fresh medium every 3 days, and split when they reached 100% confluence.

Human lung microvascular endothelial cells (HL-MVEC) were grown in complete EBM-2 medium (Lonza, Walkersville, MD, USA) at 37°C and 5% CO₂. Experiments with PLY were performed in serum-free medium, since the toxin’s activity is neutralized by cholesterol.