Hcy facilitates MKs differentiation and platelet production. A Peripheral PLT in C57BL/6J mice. Significance according to two-tailed unpaired t test (n = 8). B Representative images for staining and C quantification of mpl:eGFP⁺ cells (green) in zebrafish Tg(mpl:eGFP)smu4 larvae caudal hematopoietic tissue (CHT) region. Scale bars, 50 μm. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 10). D PLT recovery after platelet depletion by monoclonal rat anti-mouse CD42b antibody (Anti-CD42b MoAb). Significance according to two-tailed unpaired t test (n ≥ 5). E PLT in splenectomized mice. Significance according to two-tailed unpaired t test (n = 8). F PLT in C57BL/6J-Mpl^hlb219/J mice. Significance according to two-tailed unpaired t test (n ≥ 5). G Representative images for staining and H quantification of mpl:eGFP⁺ cells (green) at mpl-mutational zebrafish Tg(mpl:eGFP)smu4;mpl^smu3 larvae CHT region. Scale bars, 50 μm. Significance according to Welch ANOVA test with Dunnett T3 multiple comparisons test (n = 10). I 13 cell clusters were displayed by UMAP. Colors indicate cell types. CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEMP, megakaryocyte-erythroid-mast cell progenitor; MK, megakaryocyte. J Bar diagram showing the representative GO biological process terms of MKs subpopulations. K Bar diagram showing the percentage of each MKs subpopulation number to all cells. L Venn diagram visualizing the up-regulated biological processes of each MKs subpopulation in Hcy group compared with control group. M Representative images of PPF detected by phase contrast imaging (left) and confocal microscopy (right). β1-tubulin (green) and DAPI (blue) were stained; Scale bars, 20 μm. N Histogram showing the number of PPF-MKs. Significance according to two-tailed unpaired t test (n = 6). O Representative images and P quantification of CD41⁺ MKs (green) in mice femurs bone marrow by immunofluorescence staining. Scale bars, 50 μm. Significance according to two-tailed unpaired t test (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant

Hcy promotes MKs differentiation via GH-PI3K-Akt axis. A Venn diagram visualizing the elevated Pathway Interaction Database pathways of MKs subpopulations in Hcy group. B Heatmap showing the relative GSVA scores for each gene set based on bulk RNA-seq of BM MKs (n = 4). C and D Western blot analysis of p-PI3K and p-AKT (Ser473) in Meg-01 cells after exposure to Hcy (100 μM) for indicated time period. Total PI3K, AKT and GAPDH were used as loading control. Significance according to one-way ANOVA with LSD multiple comparisons test (n = 3). E Culture-derived MKs and F platelets were analyzed by flow cytometry. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 3). G PLT in Ghr^−/− mice. Significance according to two-tailed unpaired t test (n = 6). H Quantification of CD41⁺ MKs in femurs bone marrow of Ghr^−/− mice. Significance according to Mann–Whitney test (n = 6). I and J Western blot analysis of p-PI3K and p-AKT (Ser473) in the bone marrow MKs after exposure to Hcy (100 μM) for 30 min. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 6). K Bar diagram showing the percentage of MK2 to all cells. L Peripheral PLT. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 8). M Representative images and N quantification of CD41⁺ MKs (green) in femurs bone marrow of mice. Scale bars, 50 μm. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 8). O Bar chart showing the significantly down-regulated GO-BP terms and P PID pathways in MKs. T values are from the linear model in the limma package. Q–R Western blot analysis the level of p-PI3K and p-AKT (Ser473) in Meg-01 cells after exposure to Hcy (100 μM) with or without MT (1 μM) for 30 min. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant

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