Cd133

Western blot assay was performed as described previously 20 (link). Briefly, total protein from cells was extracted using RIPA buffer (Beyotime, Shanghai, China) supplemented with complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland). The extracted protein was separated on SDS polyacrylamide gels and transferred to the PVDF membranes. Western blotting was performed using antibodies against human COL6A1 (Abcam, Milton, Cambridge, UK), N-cadherin, E-cadherin, Vimentin, FAK, Src, p-FAK, p-Src, p-STAT1 (Tyr701), p-STAT1 (Ser727), p-STAT3 (Tyr705), STAT1, STAT3, CD133, ABC2G, SOX2, Nanog, CD63, CD9, TSG101, SOCS5, Flag, GFP, HA, Ubiqution (Ub), GAPDH, p300, c-Jun were purchased from Cell Signaling Technology (CST, USA).

CD24, CD44, CD133, Nanog, Oct4, Sox2, KLF4, c‐Myc, Gli1, Gli2, Patched1, Patched2, Smoothened, Bcl‐2, Cyclin D1, E‐cadherin, N‐cadherin, Snail, Slug and Nanog antibodies were obtained from Cell Signaling Technology (Danvers, MA). Shh protein and anti‐β‐actin antibody were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). α‐Mangostin (98% pure) was obtained from the LKT (St. Paul, MN). Accutase was purchased from Innovative Cell Technologies, Inc (San Diego, CA). Matrigel was purchased from BD Bioscience (San Jose, CA). Crystal violet was purchased from Sigma‐Aldrich (St. Louis, MO). TRIZOL was purchased from Invitrogen (Grand Island, NY). Luciferase assay kit was purchased from Promega.

RCC cell lines Ketr-3, 786-O, and ACHN as well as normal renal tubular epithelial cell HK-2 were all cultured in DMEM (high glucose) medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and streptomycin. All the cultures were maintained in humidified incubator with 5% CO₂ at 37 °C. The antibodies against β-actin, CD133, Nanog, Oct4, GAPDH, phosphorylated (Serine-311) and total NF-κB p65, PARP were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibody against LC3B was purchased from Novus (Shanghai, China), and Ki-67 was purchased from Abcam (Shanghai, China). All the secondary antibodies labeled with HRP were purchased from Beyotime (Nanjing, China). B-27 supplement (B-27) was purchased from Thermo Fisher Scientific (Shanghai, China). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from PeproTech (Rocky Hill, NJ, USA). Insulin was purchased from Sigma (Shanghai, China). Bay 11–7082 (inhibitor of NF-κB) was purchased from MCE (Shanghai, China). Sorafenib tosylate tablets were purchased from Bayer (Shanghai, China). 3-Methyladenine (3-MA, an inhibitor of PI3K) was purchased from TargetMol (Shanghai, China). YOYO-1 dye was purchased from Invitrogen (Carlsbad, CA, USA).