Western lightning plus enhanced chemiluminescence reagent

Cell lysates were separated by SDS-PAGE on 10% Tris–glycine gels. Proteins were transferred to nitrocellulose membranes and probed with antibodies against Sox2 (Merck Millipore, Billerica, MA, USA); Nestin (Novus Biologicals, Littleton, CO, USA); PDPN and β-catenin (Cell Signaling Technology, Beverly, MA, USA); N-cadherin (R&D Systems); Zeb1 (Sigma-Aldrich); STAT3 (Cell Signaling Technology); MGMT (MT3.1); Phosphorylated STAT3 (Cell Signaling Technology); and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Proteins were detected using horseradish peroxidase-conjugated IgG (Santa Cruz Biotechnology) in conjunction with Western Lightning Plus-enhanced chemiluminescence reagent (PerkinElmer, Waltham, MA, USA). Images were captured using an ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Little Chalfont, UK).

Lysates of GBM TSs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on Tris–glycine gels. Cytosolic and nuclear fractions were prepared using cytoplasmic and nuclear extraction reagents (Thermo Scientific, Rockford, IL, USA), respectively, according to the manufacturer’s instructions. Proteins were transferred to nitrocellulose membranes and probed with antibodies against cleaved capase-3, CD133, PDPN, β-catenin, active-β-catenin, CD44, snail, Cyclin D1 and cMYC (Cell Signaling Technology, Danvers, MA, USA); Sox2 (Merck Millipore, Darmstadt, Germany); Msi-1 (Abcam, Cambridge, UK); nestin (Novus Biologicals, Centennial, CO, USA); N-cadherin, Zeb1 (Sigma-Aldrich, St. Louis, MO, USA); Twist, Oct3/4, BAX, Bcl-2, PARP and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA); and FOXM1 (Bethyl Laboratories, Montgomery, TX, USA), along with Western Lightning Plus-enhanced chemiluminescence reagent (PerkinElmer, Lawrence, MA, USA). Images were captured using an ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Little Chalfont, UK). We expressed all the results of Western blot as a violin plot, and added this as a Supplementary 5. The original image of full-length blot was added as a Supplementary 6.

Cell lysates were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% Tris-glycine gels. Protein bands were transferred to nitrocellulose membranes and probed with antibodies against CD133, Nestin (Novus Biologicals, Littleton, CO, USA), PDPN and Snail (Cell Signaling Technology, Berverly, MA, USA), N-cadherin (R&D Systems, Minneapolis, MN, USA), Zeb1 (Sigma-Aldrich), Twist, Oct3/4, and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Detection was performed using horseradish peroxidase–conjugated IgG (Santa Cruz Biotechnology), in conjunction with Western Lightning Plus-enhanced chemiluminescence reagent (PerkinElmer, Waltham, MA, USA). Images were captured using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Little Chalfont, UK).