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. 2022 Dec 1;107(12):2972-2976.
doi: 10.3324/haematol.2021.280499.

Structure-function analysis of the role of megakaryoblastic leukemia 1 in megakaryocyte polyploidization

Fiona E Reed  1 , Nicole M Eskow  2 , Elizabeth Min  3 , Maximillian Carlino  4 , Rubia Mancuso  5 , Nayoung Kwon  6 , Elenoe C Smith  6 , Shannon T Larsuel  6 , Lin Wang  6 , Vanessa Scanlon  4 , Diane S Krause  7
Affiliations

Structure-function analysis of the role of megakaryoblastic leukemia 1 in megakaryocyte polyploidization

Fiona E Reed et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Effect of MKL1 on polyploidization of primary murine megakaryocytes. (A) Mean fluorescence intensity (MFI) of propidium iodide (PI) for megakaryocytes derived from wild-type (WT), Mkl1 kockout (KO), or double knockout (dKO) murine bone marrow, normalized to the MFI of the WT in each trial (dKO vs. WT: P=0.01, n=2). (B) MFI for megakaryocytes derived from WT, Mkl1 KO, or dKO murine bone marrow transduced with empty vector (EV) backbone or full-length (FL) MKL1 retrovirus. Each experiment was normalized to the MFI of the EV (WT: P=0.011, n=5; Mkl1 KO: P=0.007, n=6; dKO: P=0.028, n=4). (C) Representative ploidy peaks for WT, Mkl1 KO, and dKO marrow-derived megakaryocytes transduced with EV or FL MKL1 retroviruses, with cell number as a function of PI fluorescence. Far right: overlay of the mode-normalized ploidy peaks with EV (blue) and FL (red).
Figure 2.
Figure 2.
Investigation of MKL1 domains needed for polyploidization. (A) Schematic of domain deletion constructs (empty vector [EV] not shown): full-length (FL) MKL1, ΔRPEL, ΔBasic, ΔSAP, ΔLZ, and ΔTAD. (B) Mean fluorescence intensity (MFI) of propidium iodide for Mkl1 knockout (KO) murine bone marrow-derived megakaryocytes transduced with each deletion construct (see Figure 2A), normalized to the EV value for each experiment. Individual experiments are indicated by data point colors (FL: P=0.007, n=6; ΔRPEL: P=0.037, n=4; ΔSAP: P=0.014, n=5). (C) MFI of propidium iodide for double knockout (dKO) murine bone marrow-derived megakaryocytes transduced with each deletion construct, normalized to the EV value for each trial. Individual trials are indicated by data point colors (FL: P=0.028, n=4; ΔRPEL: P=0.0004, n=4; ΔSAP: P=0.017, n=4). (D) Summary of data, showing mean fold-change in ploidy driven by each construct relative to EV.
Figure 3.
Figure 3.
Expression of GEF-H1 in response to wild-type and mutant forms of MKL1. Representative images of megakaryocyte progenitors transduced with MKL1 deletion constructs, cultured in thrombopoietin-only medium, and immunostained for α-tubulin and GEF-H1. Tubulin was detected with mouse anti-tubulin (1:250, ThermoFisher A11126) and AlexaFluor 488-labeled donkey anti-mouse AlexaFluor 488 (1:500, LifeTechnologies A21202). GEF-H1 was detected with rabbit anti-GEF-H1 (1:50, AbCam ab155785) and AlexaFluor 555-labeled donkey anti-rabbit (1:500, LifeTechnologies A31572). Hoechst 33342 was used to identify nuclei.
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References

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