Page 153 - Haematologica March 2020

Bone marrow niche dysfunction in ET
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Figure 5. Low expression of WDR4 is correlated with enhanced proliferation, decreased senescence, and impaired differentiation of bone marrow derived mes- enchymal stromal cells (BM-MSC) isolated from patients with JAK2V617F-positive essential thrombocythemia (ET). A. WDR4 mRNA expression in BM-MSC isolated from the controls (n=20) and patients with JAK2V617F-positive ET (n=15).B. WDR4 protein expression in BM-MSC isolated from the controls (n=4) and patients with JAK2V617F-positive ET (n= 4). C–D. WDR4 shRNA and cDNA decreased or increased WDR4 expression in BM-MSC efficiently, as determined by qPCR (C) and Western blotting (D). E. WDR4 cDNA treatment decreased the proliferative capacity of BM-MSC, while WDR4 shRNA treatment increased the proliferation of BM-MSC, as measured by the CCK-8 assay. F. WDR4 cDNA increased senescence of BM-MSC as measured by β-galactosidase staining while WDR4 shRNA had the opposite effect. G. WDR4 increased the differentiation potential of BM-MSC into adipocytes, osteocytes, and chondrocytes as indicated by Oil Red O, Alizarin Red, and Alcian Blue staining, respectively. MSC used in each assay were at passage four. All the experiments (except for the quantitation of WDR4 mRNA and WDR4 protein expres- sion in clinical samples) were repeated at least three times. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Data are presented as the mean ± SD (except for mRNA expression of WDR4 in clinical samples, mean ± SEM). ET: essential thrombocythemia; CCK-8: Cell Counting Kit 8; n: number of unique donors in each group; NC: normal control; SD: standard deviation; SEM: standard error of mean.
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