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. 2020 Sep 28;8(10):385. doi: 10.3390/biomedicines8100385

Figure 2.

Figure 2

Experimental expression of Evi1 counteracts the anti-leukemic and stem cell inhibitory effects of atRA in Flt3-ITD/Npm1c-driven murine AML. (a,b) Bone marrow LCFlt3-ITD/Npm1c_vec and LCFlt3-ITD/Npm1c_Evi1 were treated with the indicated concentrations of atRA or with solvent for 48 h. (a) Cell viability was determined using metabolic activity as a proxy (Cell-Titer Glo® assay). n = 4. (b) Apoptosis was determined through Annexin V staining followed by flow cytometry. n = 2–4. (c) Myeloid differentiation. Bone marrow cells from leukemic mice were treated with 1 µM atRA or the corresponding amount of solvent for 72 h, stained with CD11b and Gr-1 antibodies, and subjected to flow cytometry. Analyses were restricted to leukemia cells (LCs) by gating on GFP-positive cells. n = 3. (d) Serial replating activity. Bone marrow LCFlt3-ITD/Npm1c_vec and LCFlt3-ITD/Npm1c_Evi1 were treated with 1 µM atRA or the corresponding amount of solvent for 72 h. A total of 3000 cells were plated per well of a six-well plate in MethoCult GF M3434. Every seven days, the numbers of colonies were quantified, and 3000 cells were re-plated. Numbers of colonies are expressed relative to those obtained with solvent-treated LCFlt3-ITD/Npm1c_vec in each round of plating. n = 3. (a–d) *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.