Gamma-secretase

(d,e) Effect of TUG1 overexpression on Nestin activity. Plasmid vectors expressing indicated genes were added to GSC-pE-Nes-222 treated with RO4929097. Phase-contrast and Nestin-EGFP images were shown in (d). Scale bars, 100 μm. (e) Intensity of Nestin-EGFP (left) and number of viable cells (right) compared with the DMSO control were quantified. Viable cells were assessed by trypan blue staining. *P<0.01, Kruskal–Wallis analysis. For all the experimental data, error bars indicate +s.d. (n=3).

Cultures were assessed in A for TRAP by enzyme histochemistry 5-8 days following exposure to Rankl. Values are means ± S.D.; n = 6 for all data sets except for Notch2Q2319X DMSO at day 5 where n = 5; the right upper panel shows representative culture fields of TRAP-stained multinucleated cells 8 days after the addition of Rankl.

Jurkat cells were treated with (D) TRAIL (20 ng/ml), MK-0752 (50 µM) or a combination of the two for 48 h. Vehicle control for (D), 0.1% (v/v) dimethyl sulfoxide. Data shown are the mean ± standard error of the mean of ≥3 independent experiments. *P≤0.05, **P≤0.01. TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.

A panel of GICs lines was treated with various concentrations of γ secretase inhibitors BMS-708163. Cells were treated with increasing concentrations of γ secretase inhibitors in triplicate wells for 72 hours, and cell viability was assessed by CellTiter-Blue assay as described in Materials and Methods. The results shown are of a single experiment with three independent replicates cell viability was measured by CellTiter-Blue assay. The graph depicts cell viability at 72 hours. Cell viability in the vehicle control was considered as to be 100%.

NSG mice were engrafted with human DND41 cells in 3 independent experiments. After DBZ treatment, marrow leukemia burden was determined by the expression of human CD45 (hCD45). Host LSK (hCD45−) (A), OB (B) and MSC frequencies (C) were determined by FACS in control (receiving culture medium) or in xenografted mice treated with vehicle or DBZ (n=8–9.group).

(E) Quantitation of lumen from A-D. (F) RNA isolated from 6d acini and expression of NOTCH1 measured by qRT-PCR in triplicate. P values are * <.01, *** <0.001.

(F) Tumour tissues were further analysed to detect the efficacy of the inhibitors by IHC staining using p-EGFR and Notch1. (4Fa) Notch1 staining was mostly confined on the cell membrane. (4Fc) After Erlotinib treatment, the Notch1 protein was cleaved and translocated to the nucleus. (4Fb) p-EGFR was overexpressed in the control group. (4Fd) Erlotinib treatment inhibited p-EGFR expression. (4Fe-f) PF-03084014 treatment reduced both Notch1 and p-EGFR expression. (4Fg-h) Synthetic therapy reduced the activation of Notch1 and EGFR pathway. Scale bar = 50 μm. *P < .05, **P < .01, ***P < .001

The levels of Aβ produced by neuronal differentiated NSCs in response to vehicle (0.1% DMSO), 10μM BSI IV, 10μM L685,458, or the indicated compounds at 3μM or 10μM for 24hours

G. In the presence of FLI-06, wound healing assay was conducted to evaluate the cell motility after transfection. Scale bar represents 200 μm. H. In the presence of FLI-06, invasion assay was conducted to evaluate the cell invasiveness after transfection. Data were from three independent experiments and were Mean ± SD. **p < 0.01 compared to cells transfected with vector, ##p < 0.01 compared to cells transfected with ACTG2. Scale bar represents 100 μm.