GBM cell culture and reagents

Both of U87 and U251 cell lines are IDH-wildtype subtype of glioblastoma (GBM-IDH-wt) according to the recent change in classification of gliomas. U87 and U251 were purchased from ATCC (Manassas, VA, USA). The authentication of the U251 and U87 cell lines have been tested by short tandem repeat (str) profiling by Department of Pathology, the Chinese University of Hong Kong in 2016 and 2017. Cells were cultured in DMEM medium (Gibco Invitrogen, Grand Island, New York, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin, at 37 °C and 5% CO₂. Cells were maintained in T75 flasks or T25 flasks, and passaged every 2–3 days when reached 70–80% confluence. Recombinant Murine Wnt3A, human R-spondin 2 and R-spondin 3 were purchased from PeproTech (Rocky Hill, NJ).