Gamma-secretase

Western blotting showing increased unconjugated SUMO1 levels in Notch1 ΔE cells treated with 10 uM DAPT for 3 days. Tubulin was used as a loading control.

HEK/APPswe cells were either left non-treated (-) or exposed to the γ-secretase inhibitor (GSI) semagacestat (+) for 19 h prior to cell harvesting and membrane preparation (Substrate accumulation). Exposure of cells with GSI prior to membrane preparation results in higher Aβ1-42 and Aβx-42 signals as compared to membranes derived from non-GSI treated cells. Direct addition of semagacestat to the membranes in the novo Aβ production assay inhibited Aβ production (Semagacestat (1 µM)). The amount of Aβx-42 is higher than Aβ1-42 suggesting that C83 is the major substrate for the Aβ42 signal detected.

HES1 expression after treatment with clinically available GSIs R04929097, BMS-708163, LY450139, and MK-0752 as determined by qRT-PCR.

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%.

Tumor spheres formation assay. Images were taken at 48 h after treating the cells with various formulations of YO (scale bar 100 um).

(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).

Western blotting showing increased unconjugated SUMO1 levels in Notch1 ΔE cells treated with 10 uM DAPT for 3 days. Tubulin was used as a loading control.

HEK/APPswe cells were either left non-treated (-) or exposed to the γ-secretase inhibitor (GSI) semagacestat (+) for 19 h prior to cell harvesting and membrane preparation (Substrate accumulation). Exposure of cells with GSI prior to membrane preparation results in higher Aβ1-42 and Aβx-42 signals as compared to membranes derived from non-GSI treated cells. Direct addition of semagacestat to the membranes in the novo Aβ production assay inhibited Aβ production (Semagacestat (1 µM)). The amount of Aβx-42 is higher than Aβ1-42 suggesting that C83 is the major substrate for the Aβ42 signal detected.

HES1 expression after treatment with clinically available GSIs R04929097, BMS-708163, LY450139, and MK-0752 as determined by qRT-PCR.

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%.

Tumor spheres formation assay. Images were taken at 48 h after treating the cells with various formulations of YO (scale bar 100 um).

(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).