Cd133

Dulbecco’s Modified Eagle’s Medium (DMEM) medium, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin/streptomycin, L-glutamine, phosphate-buffered saline (PBS) and trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), dimethyl sulfoxide (DMSO), Hoechst33342, propidium iodide (PI), cisplatin, doxorubicin and bovine serum albumin (BSA) were obtained from Sigma-Aldrich, Co. (St. Louis, MO, USA). The following primary antibodies, caspase-9 (#9502), caspase-3 (#9662), p53 (#9282), BCL-2 (#4223), BAX (#5023), AKT (#9272), phosphorylated AKT (#4060), ERK (#4695), phosphorylated ERK (#4370), Nanog (#4903), CD44 (#9502), GADPH (#5174) and β-actin (#4970), were obtained from Cell Signaling Technology (Danvers, MA, USA). CD133 (#CA1217) was obtained from Cell Applications (San Diego, CA, USA). The respective secondary antibodies, anti-rabbit IgG (#7074) and anti-mouse (#7076), were obtained from Cell Signaling Technology (Danvers, MA, USA).

CSC221, cells treated for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer^{24 (link)}. Antibodies against ALDH1 (sc-166362; Santa Cruz Biotechnology, Dallas, TX, USA), CD133 (CA1217; Cell Applications, San Diego, CA, USA), CD44 (3570; Cell Signaling Technology, Danvers, MA, USA), Lgr-5 (ab75850; Abcam, Cambridge, MA, USA), Msi-1 (ab52865, Abcam), Gli1 (sc-20687; SANTA CRUZ, Dallas, TX, USA), Gli2 (sc-271786; SANTA CRUZ), Smoothened (SMO; ab72130; Abcam, Cambridge, MA, USA), Bmi-1 (ab38295; Abcam) were used to detection. α-tubulin (2125, Cell Signaling Technology) and β-Actin (sc-47778; SANTA CRUZ) antibody was used as an internal control. The bands were cut according to the protein size region of interest before intubating with antibodies and then imaged with an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA). Uncropped images of the blot images are presented as additional data (Supple Figs. 10, 11). Bands relative density was calculated based on the density of α-tubulin and actin bands in each sample. Values were demonstrated as arbitrary densitometric units corresponding to signal intensity.

Cells treated with physciosporin for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [30 (link)]. Antibodies against ALDH1 (sc-166362; Santa Cruz Biotechnology, Dallas, TX, USA), CD133 (CA1217; Cell Applications, San Diego, CA, USA), CD44 (3570; Cell Signaling Technology, Danvers, MA, USA), Lgr-5 (ab75850; Abcam, Cambridge, MA, USA), and Msi-1 (ab52865, Abcam) were used to detect stemness factors. α-tubulin (2125, Cell Signaling Technology) antibody was used as an internal control. The luminescence photon from proteins were detected by an Image Quant LAS 4000 mini using horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) with an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA). Quantitation of bands were performed by using Multi-Gauge 3.0 software, and the relative density of each band was calculated based on the density of the internal control bands in each sample. Values are shown as arbitrary densitometric units corresponding to signal intensity. All results are representative of at least three independent experiments.