KD of miR-21 in T-ALL induces apoptosis. (A) Heatmap of the 25 most abundantly expressed miRNAs as assessed by microarray in FACS-sorted mouse CD4⁻CD8⁻ DN (n = 3) and CD4⁺CD8⁺ DP (n = 3) thymocytes compared with early stage (ES-TALL, n = 3) and late-stage (LS-TALL, n = 3) primary mouse T-ALL samples and mouse T-ALL cell line M295. (B) qRT-PCR validation of miR-21 expression in independent mouse samples compared with CD4⁺CD8⁺ DP thymocytes and normalized to sn-U6. Samples used: DN, sorted DN thymocytes; DP, sorted DP thymocytes; early T-ALL, polyclonal early stage mouse T-ALL; late T-ALL, oligoclonal late-stage mouse T-ALL samples; T-ALL cell lines, 3 independent mouse T-ALL cell lines. (C) Heatmap of the 25 most abundantly expressed miRNAs assessed by microarray in a human T-ALL cell line CUTLL1. Cells (n = 3 independent samples per condition) were treated either with dimethylsulfoxide (DMSO) or difluorophenylacetyl-L-alanyl-S-phenylglycine t-butyl ester (DAPT) (10 µM) for 3 days prior to analysis. (D) miR-21 expression analysis by qRT-PCR on human T-ALL cell lines CUTLL1, T-ALL1, DND41, and KOPTK1 (black bars, n = 3 independent samples per cell line) normalized to sn-U6 as illustrated by ΔCT values. Furthermore, 2 primary T-ALL patient samples were analyzed for miR-21 expression (gray bars). (E) qRT-PCR was performed on RNA samples extracted from the gamma secretase inhibitor (GSI)-sensitive T-ALL cell lines CUTLL1, T-ALL1, DND41, and KOPTK1. Cells were treated with DMSO or DAPT (10 µM) for 3 days. qRT-PCR for miR-21 was performed, and expression levels were normalized to sn-U6 expression. (F) Quantification of early apoptosis (7-AAD⁻ Annexin-V⁺) in fluorescein (FAM)⁺ T-ALL cell lines M295 and CUTLL1 3 days posttransfection with FAM-LNA anti-miR-21 (▪, LNA_21, n = 5) or control (□, LNA_Ctr, n = 5). Data (E-F) are pooled from 2 independent experiments. *P < .05; **P < .01; ***P < .001.