MOESM1 of R-spodin2 enhances canonical Wnt signaling to maintain the stemness of glioblastoma cells

Additional file 1: Figure S1. Potentiation effect of Rspo2 in U87 cells. A-B, U87 cells were pre-treated in serum-free medium for 24 hours, then cultured in serum-free medium containing different WNT ligands for another 24 hours. Rspo2 shows potentiation effect of Wnt3A induced β-catenin targets (A) as well as RSPO2-LGRs (B). Blk indicates U87 cells cultured in 0.1% DMSO. (C) Schematic illustration of 7TGP vector. Figure S2. The effect of Wnt3A and/or Rspos on stem cell markers in U87 and U251 cells. A, mRNA expression levels of stem cell markers in U87 cells in respond to Wnt3Aand/or Rspo2. Blk indicates U87 cells cultured in 0.1% DMSO. B, Wnt 3A and Rspo2 do not affect CD133 expression in U251 cells. U251 cells were pre-treated in serum-free medium for 24 hours, then cultured in serum-free medium containing different Wnt ligands for another 24 hours. The cells were stained with CD133-APC antibody and analyzed for CD133 positivity by flow cytometry. Blk indicates U251 cells cultured in 0.1% DMSO. Figure S3. Establishment and Characterization of U251 and U87 GSCs. A, Flow cytometry analysis of U87 and U251 GSC-like cells. B, 5000 U251 cells or GSC-like cells were seeded in GSC medium for 10 days, sphere formation was evaluated for numbers and diameters. Quantification analysis of data is expressed as the Mean ± SD from three independent experiments. C, 200 U251 cells or GSC-like cells were used for holoclone assay, where U251 GSCs show an enhanced holoclone formation ability than normal glioma cells. D–E, U87 GSCs show upregulated mRNA expression levels of β-catenin targets (D), as well as RSPO-LGR genes (E). Figure S4. Rspo2/Wnt3A prevents RA and growth factor deprivation-induced differentiation in GSCs. A, all-trans retinoic acid (10 µM RA) was used to induce differentiation in U87 GSCs for 24 hours with or without WNT ligands (20 ng/ml). Real-time PCR was used to determine the effect on differentiation. Results show that Rspo2/Wnt3A treatment rescues RA-induced U87 GSC differentiation. Blk indicates GSCs cultured in DMEM with 0.1% DMSO. B, U251 GSCs were cultured in GSC media, or GSC media without EGF and FGF, or GSC media without EGF and FGF but with Wnt3A and Rspo2 for 7 days. Phase image shows the morphology of spheres. C, real-time PCR shows that Rspo2/Wnt3A treatment abolishes the downregulation of β-catenin targets caused by growth factor deprivation. Blk indicates U251 GSCs cultured in GSC media with 0.1% DMSO. Figure S5. Wnthigh and Wntlow cell populations show different cellular behavior. A, Western blot analysis comparing the responsiveness of Wnthigh and Wntlow cell populations. B, 3000 cells/well of U251 Wnt high and Wntlow cells were pre-treated in serum-free medium for 24 hours, then cultured in serum-free medium containing different WNT ligands for another 4 days, and MTT assay was performed every 24 hours. C, Table shows serial dilution tumor inoculation assay using U251 Wnthigh and Wntlow cells.