Loss of miRNAs leads to induction of apoptosis in T-ALL cells. (A) Experimental strategy to study Dicer1 deletion in vitro. Cre^ERT was activated in the Cre^ERT transgenic murine T-ALL cell line M295^CreERT (Dicer1^lox/lox) for 3 days by addition of tamoxifen (4-OHT) to inactivate Dicer1. Control T-ALL cells (M295^CreERT) were treated with ethanol (Et-OH) only. T-ALL cells were passaged twice (P1, P2), maintained under 4-OHT-free conditions, and Dicer1 deletion was assessed by deletion PCR. (B) Dicer1 deletion PCRs on T-ALL cell line M295^CreERT after initial deletion of Dicer1 in vitro as outlined in panel A. Shown is 1 representative out of 2 performed experiments. Initial deletion after 3 days of 4-OHT treatment (left). Dicer1 deletion at day 0, 3 (P1, passage 1) and 7 (P2, passage 2) after initial Cre^ERT activation (right). (C) Experimental strategy to lineage trace Dicer1-deficient T-ALL cell populations. Both T-ALL cell lines M295 (parental) and M295^CreERT were lentivirally transduced with a loxP flanked eGFP reporter construct (M295^CreERT-GFP^lox). After administration of 4-OHT for 3 days, parental GFP expressing (GFP^hi) and Cre^ERT transgenic GFP^lox transduced GFP positive and negative (ΔMIX, GFP^hi and GFP^lo) cells were assessed by FACS for cell cycle analysis and induction of apoptosis. (D) Representative flow cytometric analysis of M295^CreERT-GFP^lox 3 days after 4-OHT treatment compared with parental M295-GFP^lox T-ALL cells. (E) Representative Dicer1 deletion PCR on DNA derived from parental M295-GFP^lox (par.), unsorted M295^CreERT-GFP^lox (Cre^ERT MIX) and FACS-sorted GFP^hi vs GFP^lo (Cre^ERT-GFP^hi vs Cre^ERT-GFP^lo) cells 3 days after 4-OHT treatment. (F) Assessment of cell cycle status of GFP^lox transduced T-ALL cells 3 days after 4-OHT treatment (M295-GFP^hi parental vs M295^CreERT-GFP^hi vs M295^CreERT-GFP^lo). (G) Quantification of early apoptosis (7-AAD⁻ Annexin-V⁺) in M295-GFP^lox (parental GFP^hi) and M295^CreERT-GFP^hi vs M295^CreERT-GFP^lo cells. Data (F-G) are pooled from 2 independent experiments. (H) Experimental approach to assess the fate of Dicer1-deleted T-ALL cells in vivo. M295^CreERT cells were treated for 3 days with 4-OHT yielding deleted and undeleted cells (ΔMIX) or Et-OH only as control (CTR), these cells were subsequently transplanted into RAG2γc^−/− recipient animals. (I) Kaplan-Meier survival plot of RAG2γc^−/− recipients receiving M295^CreERT CTR T-ALL cells or ΔMIX cells. The dashed line indicates MST: red = 16 days, green = 20.5 days. (J) Dicer1 deletion PCR on DNA from cells of the M295^CreERT T-ALL line prior to transplantation (CTR or ΔMIX, upper panel, “input”, same experiment as in panel I) and on splenocytes of RAG2γc^−/− recipient animals with late-stage T-ALL (lower panel).