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. 2020 Aug 6;5(15):e136706. doi: 10.1172/jci.insight.136706

Figure 6. ERM activates GHSR/JAK2 signaling for regulating DEL-1 expression.

Figure 6

(A) DEL1 mRNA expression determined by qPCR and DEL-1 protein levels determined by ELISA in control or GHSR siRNA-transfected HMVECs treated with ERM (10 μg/mL) or ghrelin (5 μg/mL) 3 hours (mRNA) or 6 hours (protein) (n = 6 culture sets/group). Data normalized against GAPDH mRNA are expressed as fold induction relative to ethanol (set as 1). (B) HMVECs, pretreated for 24 hours with control or GHSR siRNA (20 nM), were incubated with ERM and assayed for phosphorylation at indicated points. (C) After 1-hour pretreatment with AG490 (10 μM), LY294002 (20 μM), or SB203580 (10 μM), HMVECs were incubated 3 hours with ERM and assayed for DEL1 expression (n = 6 culture sets/group). Data normalized against GAPDH mRNA were expressed as fold induction relative to ethanol control (set as 1). (D) HMVECs were transiently transfected with hEDIL3-promoter-Luc reporter plasmid, pretreated 1 hour with inhibitors, and subsequently incubated 8 hours with ERM or control followed by luciferase assay. Data are presented as fold change relative to ethanol control, set as 1 (n = 6 culture sets/group). (E) HMVECs, pretreated as above with inhibitors, were incubated 4 hours with ERM and subjected to ChIP analysis of C/EBPβ occupancy at the EDIL3 promoter (n = 4 culture sets/group). (F) After 30-minute pretreatment with ERM or RvD1 (100 nM), HMVECs were stimulated (3 hours), or not, with IL-17 (5 ng/mL). DEL1 mRNA expression was assayed and presented as above (n = 6 HMVEC culture sets/group). (G) HMVECs were transiently transfected with hEDIL3-promoter-Luc reporter plasmid and pretreated with inhibitors. After 1 hour, the cells were treated with ERM, RvD1, or control for 30 minutes, followed by 8-hour stimulation with IL-17 and luciferase activity assay (n = 6 culture sets/group). Data are presented as fold change relative to ethanol (set as 1). (H) After 1-hour pretreatment with inhibitors, HMVECs were treated with ERM, RvD1, or ethanol for 30 minutes, followed by 4-hour stimulation with IL-17. Chromatin was immunoprecipitated with anti–C/EBPβ IgG and subjected to qPCR of the DEL1 promoter. Nonimmunoprecipitated cell extracts served as input samples. (I) After 1-hour pretreatment with inhibitors, HMVECs were incubated with RvD1 or ERM for 30 minutes and assayed for phosphorylation. In experiments shown in A–I, sequential treatments were performed without intermediate washing steps. Each compound was used at the same concentration in all experiments. Data are shown as means ± SD. **P < 0.001, ***P < 0.0001 by 1-way ANOVA with Tukey’s multiple comparisons test (A and C–H).