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.

Spontaneous migration in the “wound-healing assay.” Representative images of wound closure 16 hours after the scratch. Original magnification: ×200. Bar graphs represent migrated cells/field expressed as mean ± S.D.

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.

The expression of p-EGFR and NICD was assessed by western blot after treatment for 3 d.

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

In the presence of FLI-06 (100 nM), wound healing assay was conducted to evaluate the cell motility after transfection. Data were from three independent experiments and were Mean ± SD. values. **P < 0.01 compared to cells transfected with vector, ##P < 0.01 compared to cells transfected with ZFR. Scale bar represents 200 μm.

Effect of LY3039478 on Hes protein in SGC7901/DDP cells. A, Protein expression of Hes-1 by Western blot. B, Expression level of Hes-1 by Western blot experiment. (*p < 0.05 control with LY3039478 0 μmol/L group; **p < 0.01 control with LY3039478 0 μmol/L group).

(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.

Spontaneous migration in the “wound-healing assay.” Representative images of wound closure 16 hours after the scratch. Original magnification: ×200. Bar graphs represent migrated cells/field expressed as mean ± S.D.

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.

The expression of p-EGFR and NICD was assessed by western blot after treatment for 3 d.

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

In the presence of FLI-06 (100 nM), wound healing assay was conducted to evaluate the cell motility after transfection. Data were from three independent experiments and were Mean ± SD. values. **P < 0.01 compared to cells transfected with vector, ##P < 0.01 compared to cells transfected with ZFR. Scale bar represents 200 μm.

Effect of LY3039478 on Hes protein in SGC7901/DDP cells. A, Protein expression of Hes-1 by Western blot. B, Expression level of Hes-1 by Western blot experiment. (*p < 0.05 control with LY3039478 0 μmol/L group; **p < 0.01 control with LY3039478 0 μmol/L group).