Figure 2.
Maturation block in Mpig6bmut MKs involves reduced gene expression and TPO signaling. (A-B) Mean size distribution of WT and Mpig6bmut MKs was analyzed by flow cytometry. (A) Dot plots depicting the proportion of SSChigh MKs and the delineation between small, medium, and large MKs. (B) Values are mean ± SD (n = 6). Unpaired, 2-tailed Student t test. *P < .05; **P < .01; ***P < .001. (C) Mean surface receptor expression on the whole MK population derived from WT and Mpig6bmut mice. Values are mean ± SD (n = 4). Unpaired, 2-tailed Student t test. ***P < .001. (D) MA plot showing upregulation and downregulation of genes in native Mpig6bmut MKs derived from female mice compared with female WT control mice. Black lines point toward downregulated MK-specific genes (eg, Tubb1, Gp6, and Gp1ba). (E) Upregulation and downregulation of MK-associated genes and non-megakaryocytic blood lineage markers in native MKs from female Mpig6bmut mice compared with the respective control (n = 4). Only values with a log2-fold change <1.0 (dotted line) were considered upregulated or downregulated. Cd3d, CD3 δ chain; Csk, C-Src kinase; Fli1, friend leukemia integration 1 transcription factor; Gata1, GATA-binding factor 1; Gfi1b, growth factor-independent 1B transcriptional repressor; Hba-a1, hemoglobin subunit α; Ikzf5, Ikaros family zinc finger protein 5; Matk, megakaryocyte-associated tyrosine-protein kinase; Mpl, myeloproliferative leukemia protein; Mpo, myeloperoxidase; Runx1, runt-related transcriptions factor 1; Stat3, signal transducer and activator of transcription 3. (F) Immunostainings of femora cryosections visualizing GATA-1 expression in WT and Mpig6bmut MKs in situ. Images are representative of 10 fields of view (FOV) per mouse (n = 6). Bars represent 50 µm. (G) Quantification of phosphorylation and/or total levels of GATA-1, Jak2, STAT5a/b, and Shp2 in in vitro–differentiated starved or TPO-stimulated WT and Mpig6bmut MKs analyzed using an automated quantitative capillary-based immunoassay platform; Jess (ProteinSimple). Corresponding representative blots are shown in supplemental Figure 3F. Values are mean ± SD (n = 5). One-way analysis of variance with Sidak correction for multiple comparisons. *P < .05; **P < .01; ***P < .001. (H) Mean size distribution of WT and Mpig6b−/− MKs was analyzed by flow cytometry. Values are mean ± SD (n = 2). Unpaired, 2-tailed Student t test. *P < .05.

Maturation block in Mpig6bmut MKs involves reduced gene expression and TPO signaling. (A-B) Mean size distribution of WT and Mpig6bmut MKs was analyzed by flow cytometry. (A) Dot plots depicting the proportion of SSChigh MKs and the delineation between small, medium, and large MKs. (B) Values are mean ± SD (n = 6). Unpaired, 2-tailed Student t test. *P < .05; **P < .01; ***P < .001. (C) Mean surface receptor expression on the whole MK population derived from WT and Mpig6bmut mice. Values are mean ± SD (n = 4). Unpaired, 2-tailed Student t test. ***P < .001. (D) MA plot showing upregulation and downregulation of genes in native Mpig6bmut MKs derived from female mice compared with female WT control mice. Black lines point toward downregulated MK-specific genes (eg, Tubb1, Gp6, and Gp1ba). (E) Upregulation and downregulation of MK-associated genes and non-megakaryocytic blood lineage markers in native MKs from female Mpig6bmut mice compared with the respective control (n = 4). Only values with a log2-fold change <1.0 (dotted line) were considered upregulated or downregulated. Cd3d, CD3 δ chain; Csk, C-Src kinase; Fli1, friend leukemia integration 1 transcription factor; Gata1, GATA-binding factor 1; Gfi1b, growth factor-independent 1B transcriptional repressor; Hba-a1, hemoglobin subunit α; Ikzf5, Ikaros family zinc finger protein 5; Matk, megakaryocyte-associated tyrosine-protein kinase; Mpl, myeloproliferative leukemia protein; Mpo, myeloperoxidase; Runx1, runt-related transcriptions factor 1; Stat3, signal transducer and activator of transcription 3. (F) Immunostainings of femora cryosections visualizing GATA-1 expression in WT and Mpig6bmut MKs in situ. Images are representative of 10 fields of view (FOV) per mouse (n = 6). Bars represent 50 µm. (G) Quantification of phosphorylation and/or total levels of GATA-1, Jak2, STAT5a/b, and Shp2 in in vitro–differentiated starved or TPO-stimulated WT and Mpig6bmut MKs analyzed using an automated quantitative capillary-based immunoassay platform; Jess (ProteinSimple). Corresponding representative blots are shown in supplemental Figure 3F. Values are mean ± SD (n = 5). One-way analysis of variance with Sidak correction for multiple comparisons. *P < .05; **P < .01; ***P < .001. (H) Mean size distribution of WT and Mpig6b−/− MKs was analyzed by flow cytometry. Values are mean ± SD (n = 2). Unpaired, 2-tailed Student t test. *P < .05.

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