CDK4 and CDK6 expression and pRb phosphorylation are targets of Notch signaling in human T-ALL cell lines. (A) Whole cell extracts were prepared from human T-ALL cell lines after 7 days of in vitro GSI or DMSO treatment to analyze differences in CDK4 (top) and CDK6 expression (middle). GAPDH (bottom) was used as a loading control. Band intensities of protein expression were normalized to GAPDH and represented graphically using ImageJ software, version 1.31, supported by Wayne Rasband (NIH). (B) Immunoblot of whole-cell lysates prepared from human T-ALL cell lines that were treated with GSI or DMSO for 7 days to analyze differences in pRb phosphorylation, pRB Ser780 (top panels), pRb Ser795 (middle panels), and GAPDH (bottom panels; loading control). (C) Graphical representation of relative G₁ rescue indices. Human T-ALL cell lines DND-41, HPB-ALL, and T-ALL1 were retrovirally infected either with MSCV-IRES-YFP and MSCV-IRES-DsRed II (vector, black bar), MSCV-cyclin D3-IRES-YFP and MSCV-CDK4-IRES-DSRed (CDK4 + D3, gray bar), or MSCV-cyclin D3-IRES-YFP and MSCV-CDK6-IRES-DSRed (CDK6 + D3, white bar). The 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 the GSI-induced G₁ arrest in the uninfected cell line.