Page 155 - Haematologica March 2020

Bone marrow niche dysfunction in ET
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Figure 7. WDR4 acts through the ERK–GSK3β–CREB pathway to enhance IL-6 expression and secretion by bone marrow derived mesenchymal stromal cells (BM- MSC). A. Graphic representation of the quantification of 12 proteins with the most significant difference in phosphorylation status between MSC infected with LV- shWDR4 and MSC in the control group, as measured by a phospho-kinase array of 43 phosphorylated kinases. B. Western blot analysis of phosphorylation levels of GSK3β (S9), AKT1/2/3 (S472/S473/S474), ERK1/2 (T202/Y204, T185/Y187), and CREB (S133) in MSC infected with LV-shWDR4 or LV-WDR4 and their respective controls. C. CREB-specific siRNA decreased CREB expression in BM-MSC efficiently, as determined by qPCR and Western blotting. D–F. IL-6 induction by WDR4 over- expression was at least partially suppressed by an ERK1/2 inhibitor (SCH772984), GSK3 inhibitor (SB216763), or CREB-specific siRNA as determined by qPCR (D), Western blotting (E), and ELISA (F). MSC used in each assay were at passage four. All the experiments 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. qPCR: quantitative real-time polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; NC: normal control; SD: standard deviation.
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