Fig 1: MKL1 involvement in Dyrk control of Mk morphogenesis.(A) Effects of Dyrk1 inhibition on targets of MKL1 and P-TEFb. Cord blood CD34+ cells cultured for 6 days in Mk medium with or without 5 µM inhibitors underwent immunoblot (IB) of whole cell lysates. Arrows indicate Filamin A isoforms. (B) Tubulin-normalized densitometry signals from IBs as in (A). Graph shows mean fold changes with inhibitors ± SEM for 3 independent experiments. *P < 0.05; **P < 0.01, 1-way ANOVA with Tukey’s post hoc test. (C) Transcriptomic effects in human mK precursors of ontogenic stage (adult versus neonatal) and Dyrk inhibition (neonatal with or without inhibitors). CD34+ cells cultured for 4 days in Mk medium underwent purification of CD61+ cells followed by RNA-Seq. Overlapping genes (Sh) with hypergeometric P values are shown; n = 3 independent experiments. See Supplemental Table 1 for gene lists. (D–F) MKL1 requirement for morphogenesis enhancement. Marrow progenitors from indicated strains cultured for 3 days in murine Mk medium with or without 5 µM harmine underwent flow cytometry after costaining with FITC-anti-CD41 and PI. (D) Mk polyploidization (PI). (E) Graph shows relative percent Mk = 8N ± SEM, n = 4/group. ***P < 0.005, 2-way ANOVA. (F) Mk size (FSC). Graph shows mean ± SEM, n = 4/group. *P < 0.05, 2-way ANOVA. (G) MKL1 localization. Human CD34+ progenitors cultured 24 hours in Mk medium with or without 5 µM inhibitors underwent immunofluorescent staining (IF) and confocal microscopy (Zeiss LSM700, original magnification, ×630; scale bar: 10 µm). (H) Graph shows mean ratio nuclear/cytoplasmic MKL1 signal ± SEM for 3 independent experiments. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test.