Overexpression of the miR-21 target gene PDCD4 induces apoptosis in T-ALL. (A) Box plot expression analysis of PDCD4 on RNA extracted from fractionated BM aspirates of 174 T-ALL patients and 73 normal controls (left panel), expression data publicly accessible through Leukemia Gene Atlas⁴⁰  and on RNA extracted of FACS-sorted CD3⁺CD4⁺CD8⁺ primary human thymocytes (n = 4) compared with primary T-ALL specimens (n = 15), expression data publicly accessible through gene expression omnibus (GEO) data set browser (GSE62006³⁵ ) (right panel). (B) Pdcd4 qRT-PCR performed on FAM⁺ mouse M295 and human CUTLL1 T-ALL cells after FAM-LNA-mediated miR-21 KD (or control transfection). Data were pooled from 2 independent experiments. (C) Western blot analysis for PDCD4 in M295 and CUTLL1 T-ALL cells was performed 3 days p.tr. T-ALL cells were transfected with FAM-LNA-Ctr or FAM-LNA-21 for miR-21 KD, and protein lysates of FACS-sorted cells were assessed for PDCD4 expression. (D) Overexpression of PDCD4 in T-ALL. A lentivirus-based system was employed to overexpress human PDCD4 in T-ALL cells. PDCD4 and eGFP contain 2 independent PGK promoters (upper panel). Experimental protocol: T-ALL cells were transduced with control virus (pL-+-eGFP) or PDCD4 overexpression virus (pL-PDCD4-eGFP) and analyzed for apoptosis induction 3 days following transduction (lower panel). (E) T-ALL cell lines M295 and CUTLL1 strongly induce early apoptosis (eGFP⁺, 7-AAD⁻ Annexin-V⁺ cells) after 3 days in PDCD4 transduced cells compared with control. (F) Double KD experiment to simultaneously downregulate miR-21 and Pdcd4 to quantify apoptosis induction in T-ALL cells. T-ALL cells were cotransfected with Cy5-coupled siRNAs (Ctr or a mix of 2 independent siRNAs targeting human or mouse Pdcd4) and FAM-coupled LNAs (Ctr or miR-21). Three days p.tr., Cy5⁺FAM⁺ cells were assessed for apoptosis induction (G). Cy5⁺FAM⁺ T-ALL cells generated through the experimental protocol in panel F were assessed for early apoptosis (7-AAD⁻ Annexin-V⁺ cells). miR-21 and Pdcd4 double KD cells were compared with cells that were transfected only with miR-21 KD (LNA-21 and siRNA-Ctr) or transfected with control oligonucleotides (LNA-Ctr and siRNA-Ctr) and cells where only Pdcd4 was downregulated (LNA-Ctr and siRNA-Pdcd4). Data (B, E, G) are pooled from 2 independent experiments. *P < .05; **P < .01; ***P < .001.