TLX3 knockdown correlates with reduced expression of miR-125b and slows proliferation of T-ALL. (A) Flowchart of TLX3/KD and 125/KD experiments. A lentivirus-based system was used to downregulate TLX3 or miR-125b in T-ALL cells. CTRL eGFP and mCherry vectors express, respectively, irrelevant shRNA and “sponge” RNA. Sorted transduced T-ALL cells were cocultured with MS5-DL1 stromal cells. The DND41 cell line was grown in stroma-free standard culture conditions for functional and molecular analyses. (B) Western blot analysis of TLX3 silencing. Two TLX3/KD vectors efficiently silenced TLX3 protein in DND41 cells (left panel), of which one (right panel) repressed TLX3 in primary T-ALL. (C) miR-125b expression in TLX3/KD vs CTRL in DND41 cells and in primary T-ALLs. Results of R-Q-PCR analyses performed in (n = 7) independent experiments for DND41 cells and in (n = 3) independent T-ALL samples. (D-E) In vitro cell growth of TLX3/KD cells: (D) DND41 cell line (n = 3) and (E) primary T-ALL (n = 1) made with 4 technical replicates. Data represent the mean ± standard error of the mean (SEM) of 3 experiments for DND41 and of 4 technical replicates for T-ALL. (F) Ectopic expression of miR-125b rescues growth inhibitory effect of TLX3 knockdown. DND41 cells were doubly transduced in the following conditions: (CTRL): CTRL/eGFP and CTRL/mCherry that do not express any miRNA; (TLX3/KD): CTRL/mCherry and TLX3/KD(eGFP); (125b/OE): CTRL/eGFP and 125b/OE(mCherry); (TLX3/KD+125b/OE): TLX3/KD(eGFP) and 125b/OE(mCherry). Transduced cells were sorted and cultured in standard conditions. GFP/mCherry DP cells were enumerated by flow cytometer; (n = 3) independent transduction experiments. (G) Reduced leukemic propagation of TLX3/KD DND41 cells in mice. Shown are engraftment levels in spleens 6 weeks after transplantation. Bars represent median values (n = 2). (H) In vitro growth of 125b/KD DND41 cells (n = 3). Data represent the mean value ± SEM. *P < .05, **P < .01, ***P < .001, Mann-Whitney U test.