Cyclin D3 is a target of Notch signaling in human T-ALL cell lines. (A) Cell-cycle analysis was performed on human T-ALL cell lines DND-41, HPB-ALL, and T-ALL1 that were treated with 0.1% DMSO (top panels) or GSI, 3 μm IL-CHO (bottom panels) for 7 days. FACS plots are representative of results of 3 independent replicates. Graphical representation of the mean (± SE) of percentage of cells in G₁ phase of cell cycle under the 2 treatment conditions for the 3 experiments conducted. Two-tailed Student t tests were performed using GraphPad Prism version 4.0 for OS X (GraphPad Software, San Diego, CA) **P < .05.. (B) Whole cell extracts were prepared from human T-ALL cell lines after 7 days of GSI or DMSO treatment to analyze difference in cyclin D3 (top) and Notch1 expression (middle). GAPDH (bottom) was used as a loading control. (C) Graphical representation of relative G₁ rescue indices. Human T-ALL cell lines were retrovirally infected with either MSCV-IRES-YFP (vector, open bar) or MSCV-cyclin D3-IRES-YFP (cyclin D3, solid bar). Infected cells were sorted for enrichment and treated with DMSO or GSI for 7 days. Cell-cycle analysis was performed to determine the percentage of cells arrested in G₁ with GSI-treatment, compared with DMSO. Relative G₁ rescue indices were calculated for each cell line after normalizing against GSI-induced G₁ arrest in the uninfected cell line.