A 21427

Cells were seeded on gelatin-coated circular micro cover glass (d = 18 mm, Matsunami, Japan) submerged in a 60-mm dish (TPP, Switzerland). After 7 days of treatment with/without 200 µM CoCl₂, cells were washed with phosphate buffered saline (PBS) 3 times and then fixed with 4% paraformaldehyde (Nacalai-Tesque) at room temperature for 20 min followed by blocking with 10% FBS in PBS containing 0.05% Tween 20 (Nacalai-Tesque) for 1 h at room temperature. The cells were then incubated with anti-CD34 rabbit polyclonal antibody (H-140) (1:50, sc9095, Santa Cruz, CA) or anti-mouse TER-119/erythroid cells rat monoclonal IgG (1:100, 116,201, BioLegend, CA) diluted with blocking solution overnight at 4 °C. After PBS washes, anti-rabbit IgG linked with Alexa Fluor 555 (1:1000, A21427, Invitrogen, CA) was used to detect anti-CD34 antibody and anti-rat IgG linked with Texas red (1:1000, T6392, Invitrogen) was used to detect anti-TER-119 antibody. Incubation with the secondary antibody was performed for 1 h at room temperature. After PBS washes, the samples were prepared on glass slides (Matsunami) using VECTASHIELD Mounting Medium with 4', 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, CA). Images were taken using an inverted light microscope equipped with a fluorescence light device (IX-80, Olympus, Japan).

Cells (1 × 10⁶) were washed twice with HEPES-buffered saline, treated with Accutase (ThermoFisher, A1110501), and fixed for 20 minutes at room temperature in 2.0% PFA. Cell membranes were permeabilized by adding 0.1% TritonX-100 in PBS for 20 minutes at room temperature. Cell pellets were collected and incubated with primary antibodies diluted in blocking solution: mouse anti-Olig2 (1:200; Merck Millipore, MAPN50), mouse anti-beta III tubulin (1:150; Merck Millipore, MAB1637), mouse anti-GFAP (1:200; Merck Millipore, MAB360), rabbit anti-TTR (1:300;; ABBIOTC, 250892) and incubated overnight at 4 °C. After washing, secondary antibodies anti-rabbit Alexa Fluor-488 (Invitrogen, A-11008) or anti-mouse Alexa Fluor-555 (Invitrogen, A-21427;), were diluted in PBS and incubated for 1.5 hours at room temperature. Flow cytometric evaluation was conducted within 5 minutes. Cells were analyzed by flow cytometry using an EPICS XL-MCL FACScan (Becton–Dickinson, Mountain View, CA, United States).

The primary antibodies α-NS3 GTX12452(1 : 200), α-E s4G2 clone Hb119 (1 : 200), and the secondary antibodies rabbit α-mouse AF555 (A-21427, Invitrogen), goat α-rabbit AF594 (A-11012, Invitrogen) were diluted in a PermWash solution. Primary antibodies α-GAPDH (1 : 4000) (A01622, Genescript), α-Flavivirus Glycoprotein (1 : 1500) (Ab214336, Abcam), α-p24 (1 : 1000) (ab9044, Abcam), α-VSV-Glycoprotein (VSV-G) (1 : 1000) (V-5507, Sigma) and secondary α-goat IgG HRP (1 : 4000) (A8919, Sigma), α-mouse immunoglobulins HRP (P0260, Dako) were diluted on 10 % Non-Fat-Dried Milk in 1X PBS 0.1 % Tween.