Specific inhibition of JAK2 V617F-driven activation by modulation of hTpoR conformations in primary BMCs. (A-D) Colony-forming unit megakaryocytes (CFU-Mk) assays. Lineage-negative bone marrow cells were isolated from Mpl KO JAK2 WT/WT or JAK2 WT/V617F mice (n = 4) and retrovirally transduced with an empty vector, hTpoR WT, or the indicated mutants. One day after infection, the cells were plated in a semisolid collagen-based megakaryocyte culture medium with lipids supplemented with 10 ng/mL of mIL-3 and 20 ng/mL of hIL-6. In the condition with Tpo (C-D), 50 ng/mL of hTpo was added. Megakaryocytes were stained with acetylthiocholine iodide after 7 days and counted blindly with an inverted microscope. The colonies were separated into small (3-20 cells), medium (21-49 cells), and large (>50 cells). Values represent the mean (±SD) of 4 independent experiments with 2 different transductions. Statistics: two-way analysis of variance followed by the SIDAK multiple comparison test. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗P < .05. (E) Illustration of the single-cell colony assay experimental protocol. Lineage-negative bone marrow cells were isolated from Mpl KO JAK2 WT or JAK2 V617F mice and retrovirally transduced with hTpoR-IRES-GFP or an empty vector. Twenty-four hours after infection, stably transduced LSK cells were sorted by flow cytometry and plated at 1 cell per well (n = 32 per condition) in 96-well plate with MethoCult medium with SCF alone, SCF + Tpo, or SCF + Epo + IL-6 + IL-6. The presence of CFU was assessed after 10 days. (F) Number of single-cell colony-forming unit from LSK cells expressing indicated constructs in Mpl KO JAK2 WT or Mpl KO JAK2 V617F background.