P glycoprotein

Spheroids were transferred to ULA plates, washed three times in PBS and fixed with 4% PFA for 1 hour at 4 °C. Spheroids were washed again then permeabilized with 0.5% Triton X-100 in Tris-Buffered Saline with 0.05% Tween20 (TBST) overnight at 4 °C and then blocked with 0.1% Triton X-100/3% BSA in TBST for 2 hours at room temperature (RT). Primary antibodies Multidrug resistance protein-2 (MRP2-Abcam) and P-glycoprotein (Pgp-Abcam) were diluted 1 : 20 in 0.1% Triton X-100/1% BSA in TBST were incubated with the spheroids overnight at 4 °C. Spheroids underwent three 1 hour washes with 1% Triton X-100 in TBST then incubated with secondary Alexa Fluor 488 Donkey Anti-Mouse antibody (Life Technologies) diluted 1 : 1000, Hoechst diluted 1 : 5000 and Phalloidin 568 diluted 1 : 250 in 0.1% Triton X-100/1% BSA in TBST overnight at 4 °C. Spheroids were finally washed for 1 hour then mounted with Prolong Gold (Life Technologies) onto a glass microscope slide. Maximum intensity projection images of spheroids were taken using a Zeiss Axio Observer microscope with Apoptome using 40× oil objective.

hCMEC/D3 were obtained from Dr. Peter Couraud^{59 (link)} INSERM, France. WntC59 was purchased from AdooQ Biosciences (Irvine, CA). Human Recombinant Wn3a, and GF120918 (GF) were purchased from R&D Systems (Minneapolis, MN). Rhodamine 800 and Rhodamine 123 were purchased from Sigma (St. Louis, MO). IRDye 800 CW PEG were purchased from LICOR (Lincoln, NB). EBM-2 media was purchased from Lonza (Walkersville, MD). The E-plates for RTCA were obtained from ACEA Biosciences (San Diego, CA). Transwell and tissue culture plates were purchased from Corning (Tewksbury, MA). Trizol and tetramethylrhodamine BSA was purchased from Life Technologies (CA, USA). P-glycoprotein and Claudin-1 antibodies were purchased from Abcam (Cambridge, MA). β-catenin, claudin-5 and β-actin antibodies were purchased from Sigma (St. Louis, MO). Human fetal brain total RNA was purchased from Takara Bio USA, Inc. (Madison, WI). Primers were obtained from Invitrogen (CA, USA) and primer sequence information is provided in Table 1 Supplementary information.