β_c and/or β_IL3 receptor signaling enhances megakaryopoiesis in Cul5-deficient mice. Numbers of (A) platelets (n = 5), (B) megakaryocytes per field from histological sections of sternal BM (n = 5), and (C) HSC (Lin^–Sca1⁺Kit⁺CD150⁺CD48^–, n = 5, left panel), CD41⁺HSC (n = 5, center panel) and percentage CD41⁺HSC in the HSC population (n = 5, right panel) in Cul5^fl/flVavCre, Cul5^fl/fl, β_c^–/–β_IL3^–/–Cul5^{+/+ or fl/fl} and β_c^–/–β_IL3^–/–Cul5^fl/flVavCre, mice. Fold-differences in numbers of platelets, megakaryocytes, HSC and CD41⁺HSC between Cul5^fl/flVavCre and Cul5^fl/fl mice or β_c^–/–β_IL3^–/–Cul5^fl/flVavCre and β_c^–/–β_IL3^–/–Cul5^{+/+ or fl/fl} mice are indicated above the bar graphs. (D) Numbers of MPP (Lin^–Sca1⁺Kit⁺CD150^–CD48^lo/–), HPC1 (Lin^–Sca1⁺Kit⁺CD150^–CD48⁺), HPC2 (Lin^–Sca1⁺Kit⁺CD150⁺CD48⁺), HPC1Flt3^hi and HPC1Flt3^lo cells (upper panel, n = 5), and CMP (Lin^–Sca1^–Kit⁺CD34⁺FcgRII/III^lo), GMP (Lin^–Sca1^–Kit⁺CD34⁺FcgRII/III⁺), MEP (Lin^–Sca1^–Kit⁺CD34^–FcgRII/III^–) and CFU-E (Lin^–Sca1^–Kit⁺CD150^–Endoglin^hiFcγRII/III^lo) (lower panel, n = 4) in Cul5^fl/flVavCre, Cul5^fl/fl, β_c^–/–β_IL3^–/–Cul5^{+/+ or fl/fl} and β_c^–/–β_IL3^–/–Cul5^fl/flVavCre, mice. Each point is data from an individual mouse, bars represent mean ± SD. ∗P < .05, ∗∗P < .01 for comparison of Cul5^fl/flVavCre and β_c^–/–β_IL3^–/–Cul5^fl/flVavCre data with their respective Cul5^{+/+ or fl/fl} controls and Cul5^fl/flVavCre with β_c^–/–β_IL3^–/–Cul5^fl/flVavCre (1-way ANOVA with Dunnett’s correction for multiple comparisons). (E) Proliferation of Cul5^fl/flVavCre and Cul5^fl/fl LSK cells in liquid cultures stimulated with IL-3 or GM-CSF, assessed after 3 days using CellTitreGlo. Each line represents data from a single mouse. (F) Analysis of phospho(p)Stat5 induction by mass cytometry in LSK cells from Cul5^fl/flVavCre and Cul5^fl/fl mice. Each line shows mean ± SD of 3 independent samples pooled from multiple mice of each genotype stimulated with 1000 units/mL IL-3 or GM-CSF for the times indicated. ∗P < .05 for comparison of data from Cul5^fl/flVavCre with Cul5^fl/fl at each time point (paired t test with Holm-Sidak correction for multiple comparisons). min, minutes; RLU, relative light units.