Dapt gsi 9

The human glioma stem/progenitor cell line SU3 and nude mice expressing EGFP (NC-CB57/6J-EGFP) were prepared by our laboratory [4 (link), 5 (link)]. The remaining reagents were purchased from companies as follows: rat C6 glioma cell line (Shanghai Institutes for Biological Sciences), RFP transgenic kit (Genechem, Shanghai), γ-secretase inhibitors DAPT (GSI-IX) (Selleck), Dulbecco's Modified Eagle's Medium (Gibco), fetal bovine serum (Hyclone), Caspase—Glo 3/7 assay kit (Promega), Trizol solution (Invitrogen), reverse transcription kit (Fermentas), ECL chemiluminescence reagent, trypan blue, GAPDH antibody (Biyuntian, Shanghai), Notch-1, NICD, Bcl-2 and pAKT antibodies (Cell Signal), 2', 3'-cyclic nucleotide 3' phosphodiesterase (CNP) monoclonal antibody (Abcam), qPCR apparatus and SYBR Green qPCR Mix (BioRad), apoptosis kit (BD), flow cytometry instrument (Beckman), inverted fluorescence microscope (Carl Zeiss), linear accelerator (Siemens Primus), and in vivo fluorescence imaging system (Maestro EX, CRi).

O4⁺-purified cells and LGG275 cells were infected with either a control-YFP or NICD-YFP virus (gift from Dr. Sutton, Yale, New Haven, CT, USA; multiplicity of infection = 6). NICD cDNA codes for amino acids from 1762 to 2556 of human NOTCH1 [24 (link)]. Five days after transduction, RNAs were extracted using the Arcturus Picopure RNA kit (Thermo Fisher), and 100–200 ng of cDNA was synthesized with random primers and reverse transcriptase (Promega, Charbonnières-les-Bains, France, GoScript). Quantitative PCR was performed using 2.5 ng of cDNA in duplicates. Primers (Sigma) are listed in Table S3. The KAPA SYBR PCR kit (Sigma) was used for QPCR (Light Cycler 480, Roche). Gene expression was calculated using the 2^−ΔΔCt method, using β-actin (ACTB) for normalization. For inhibitor experiments, LGG275 cells were treated with γ-secretase inhibitors (DAPT (GSI-IX, Selleck Chemicals, Breda, Netherlands) and LY411575 (Peprotech)) at 10 µM for 5 days before RNAs were extracted for QPCR. For the DLL4 ligand experiment (Figure S13A), plates were coated with DLL4 (5 µg/mL; Peprotech) or BSA at 4 °C overnight before seeding the cells. RNAs were extracted after 4 days. For BMP signalling experiments described in Figure 6C, cells were treated with 10 ng/mL of BMP2 or BMP4 (Peprotech) for 5 days with the addition of new cytokines every 2 days.

γ-secretase inhibitor DAPT (GSI-IX) was purchased from Selleckchem (Cat No. 208255-80-5, Houston, TX, USA), IL-1 receptor antagonist Anakinra was purchased from TOCRIS (Cat No. 185413-30-3, Minneapolis, MN, USA) and the leptin receptor antagonist, Allo aca, was purchased from Peptides international (Cat No. PCS-32627-PI, Louisville, Kentucky, USA). The DAPT was dissolved in 4% DMSO/Safflower Oil, Anakinra and the Allo aca were dissolved in 10% DMSO/0.9% NaCl. Before use, all of the drugs were freshly dissolved.