Bms 708163

The four glioma cell lines used in this study were obtained from the JCRB cell bank. The eight glioma initiating cell lines were maintained in neurosphere medium by using a previously described method [29 (link)] to isolate neurosphere-forming cells from surgical specimens of human GBM. The study was approved by the Institutional Review Board of Toho University (H22-62). These GIC lines were cultured as GBM neurospheres in DMEM/F12 medium supplemented with B27 (Invitrogen, Grand Island, NY, USA), L-glutamine (GIBCO), penicillin/streptomycin and growth factors (20 ng/mL EGF and 20 ng/mL FGF-2; Invitrogen). DAPT, BMS-708163, RO4929097 and NVP-BKM120 were purchased from Selleck Chemicals. For in vitro use, all inhibitors were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to a concentration of 10 mmol/L, stored at −20 °C and further diluted to an appropriate final concentration in DMEM/F12 medium at the time of use. The DMSO in the final solution did not exceed 0.1% (v/v).

We purchased CMK (HY-52101) from MedChemExpress, and etoposide (S1225), BMS536924 (S1012), BMS708163 (S1262), DMOG (S7483) and rapamycin (S1039) from Selleckchem. The effects of the drugs on cell proliferation were determined using a CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay kit (Promega) according to the manufacturer’s instructions. We plated 3000 cells in each assay of 96-well plates. One day later, we treated cells with a range of drug concentrations prepared by serial dilution plus 200 uM rapamycin or dimethyl sulfoxide (DMSO; four replicates per condition). The plates were incubated at 37 °C and 5% CO₂ (v/v). After 2 days, 20 μl of the CellTiter 96® AQ_ueous One Solution Reagent was directly added to the culture wells, and incubated for 1 h, and then the absorbance was recorded at 490 nm with Epoch (Biotek). The relative growth was normalized to the untreated samples in each group.

Rabbit monoclonal anti-ZEB1 (3396, s; clone D80D3), cleaved NOTCH1 (4147, s; clone D3B8; specific for detecting NICD1), RBPJ (5313, s; clone D10A4), E-cadherin (3195, s; clone 24E10), Vimentin (5741, s; clone D21H3), Tubulin (2125, s; clone 11H10), pERBB3 (2842, s; clone D1B5) and ERBB3 (12708, s; clone D22C5) were purchased from Cell Signaling and used at a 1:3,000 dilution for western blotting. Goat polyclonal anti-Actin (sc-1616) was purchased from Santa Cruz and used at a 1:1,000 dilution for western blotting. Rabbit polyclonal anti-NOTCH1 (ab27526) was purchased from Abcam and used at a 1:3,000 dilution for western blotting. Mouse monoclonal anti-FLAG (F1804; monoclonal; clone M2) was purchased from Sigma and used at a 1:5,000 dilution for western blotting. siRNAs were purchased from Santa Cruz (pooled siRNAs) or Origene (individual siRNAs for ZEB1 and RBPJ). All short hairpin RNAs and chemicals were purchased from Sigma, except for BMS-708163 and gefitinib, which were purchased from Selleckchem. Control GFP (LPP-EGFP-LV105-025) and ZEB1 lentiviral particles (LPP-F0876-Lv105-200-S) were purchased from Genecopoiea.