Cellular and transcriptional effects of HES1 knockdown in human T-ALL cells. (A) Western blot analysis of HES1 expression in CUTLL1 T-ALL cells transduced with shRNAs targeting the Renilla luciferase gene (shLUC) or HES1 (shHES1). (B) Quantification of cell growth in CUTLL1 cells expressing shLUC or shHES1. (C) Cell cycle analysis of CUTLL1 cells expressing shLUC or shHES1. (D) Representative flow cytometry plots after annexin V-allophycocyanin (annexin V-APC) and 4′,6-diamidino-2-phenylindole (DAPI) staining and quantification of apoptosis in CUTLL1 cells expressing shLUC or shHES1. Percentage populations are indicated in each quadrant. (E) Heat map representation of the top 50 differentially expressed genes (P < .01) between shLUC and shHES1 CUTLL1 cells. The scale bar shows color-coded differential expression, with red indicating higher levels and blue indicating lower levels of expression. (F) Representative examples of gene set enrichment plots corresponding to GSEA analysis of MSigDB C2 data sets enriched in the expression signature associated with shHES1 CUTLL1 cells compared with shLUC controls. (G) GSEA analysis plot of genes upregulated on HES1 shRNA knockdown in the expression signature associated with GSI-induced NOTCH1 inhibition in CUTLL1 cells. Bar graphs indicate mean values of triplicate measurements, and error bars represent standard deviation.