Ro4929097

Two-cell or morula stage embryos were cultured in drops of M16 medium (Sigma) covered with mineral oil (NidOil, EMB) at 37°C, 5% CO₂, containing the corresponding pharmacological inhibitor or only DMSO as control until the corresponding stage. The following inhibitors and concentrations were used: 10 or 20 μM of the γ-secretase inhibitor RO4929097 (S1575, Selleckchem) (Münch et al., 2013 (link)) and 10 μM of the TEAD/YAP inhibitor Verteporfin (SML0534, Sigma) (Liu-Chittenden et al., 2012 (link)).

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).

Human NSCLC cells HCC827 (Cat no. TCHu73) and human embryonic kidney 293T cells (Cat no. SCSP-502) were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The Erlotinib resistant HCC827 cell line was defined as HCC827/ER cells. HCC827/ER cells with acquired resistance to Erlotinib were obtained from the Key Laboratory of Oncology, Chongqing Cancer Institute. The HCC827 and 293T cells were cultured in DMEM (HyClone, Cat no. SH30243.01B) supplemented with 10% FBS (BI Biotech, Cat no. 04-001-1A). The HCC827/ER cells were maintained in 10% FBS DMEM supplemented with 1–5 µM Erlotinib. All cells were cultured at 37°C in a humidified incubator containing 5% CO₂. Erlotinib (Cat no. S7786), MK-2206 (Cat no. S1078), and RO4929097 (Cat no. S1575) were obtained from Selleck Chemicals; (Houston, TX, U.S.A.). To prevent the effects of Erlotinib, the HCC827/ER cells were cultured in a normal medium for ≥2 weeks before their use in further experiments.

The following primer sequences were used: OGT forward: CAGCATCCCAGCTCACTT, reverse: CAGCTTCACAGCTATGTCTTC; HES1 forward: ACGTGCGAGGGCGTTAATAC, reverse: GGGGTAGGTCATGGCATTGA; OGA forward: CGAGTGAACATTCCCATCACT, reverse: CCCAAAGGAGCACAGATGTT; PTEN forward: CGAACTGGTGTAATGATATGT, reverse: CATGAACTTGTCTTCCCGT.
The following antibodies from Cell Signaling Technology (Danvers, MA) were used: anti-OGT (#5368), anti-HES1 (#11988), anti-phospho-Ser235/236-S6 (#4858), anti-γ-H2AX (#9718), anti-PARP (#9542), and anti-Histone 3 (#4499). Mouse-derived anti-O-Linked N-Acetylglucosamine (RL2) (ab2739) was acquired from Abcam (Cambridge, UK). Mouse anti-α-tubulin (#CP06) was from Merck Millipore (Burlington, MA).
The following chemical compounds were used: OSMI1 compound was kindly provided by Professor Suzanne Walker (Harvard Medical School). Additional OSMI1 compound was purchased from Sigma Aldrich (SML1621). Efficacy of the two OSMI1 stocks was determined to be similar (data not shown); O-GlcNAcase inhibitor PUGNAc (A7229), MG132 proteasome inhibitor (M7449), mTORC1 inhibitor Everolimus (Ev) (SML2282) and PR inhibitor mifepristone (MF) (M8046), were purchased from Sigma Aldrich. γ-secretase inhibitor (GSI) RO4929097 was purchased from Selleckchem (S1575).