Fig. 3.

Cmpk2 is identified as Gata6 direct downstream target gene in ECs for the regulation of monocyte adhesion, migration and pro-inflammatory macrophage formation. (A–C) The expression of Cytidine/uridine monophosphate kinase 2 (Cmpk2) by RT-qPCR (A) and Western blot (B) with quantification data on the right in aortic ECs of Gata6^ECKO;Apoe^KO hyperlipidemic and Apoe^WT mice (n = 5), and in oxLDL stimulated HAECs of GATA6-siRNA knockdown and scramble siRNA control by RT-qPCR (C) (n = 5). (D) Sequence analysis of GATA6 binding sites (red oval) in the proximal promoter region of CMPK2 gene. (E) Chromatin immunoprecipitation (ChIP) assay of GATA6 and the promoter of CMPK2 gene with gel images on the top. (F) The luciferase activity of proximal-2kb Cmpk2 promoter with Gata6 binding sites by Gata6 expression vector in 293T cells (n = 3). (G–H) The expression of NLRP3 inflammasome components, NLRP3 and IL-1β, in oxLDL-treated ECs by RT-qPCR (G) and Western blot (H) with quantification data on the right, and the effects of GATA6-siRNA, CMPK2 vector for overexpression or the combination on NLRP3 inflammasome activation. (I) Secreted IL-1β level in conditioned medium of GATA6-siRNA or scramble-siRNA ECs by ELISA (n = 5). (J–L) The quantitative data of monocyte adhesion to GATA6-siRNA or scramble-siRNA ECs (J), monocyte migration in Boyden chamber assay (K) and macrophages engulf of Dil-labeled oxLDL (L) after treatment of condition medium from GATA6-siRNA or scramble-siRNA ECs (n = 5). Quantification of Western blot was carried out with Image J and normalized to loading control β-actin. Unpaired 2-tailed student t-test for A-C, G-L and nonparametric statistical test for F.