Figure 2: C/EBPβ regulates Del-1 expression in HUVEC.

HUVEC were transfected with control siRNA, siRNA to C/EBPβ, control vector or with C/EBPβ expression vector. (a) After 24 h, the cells were lysed and whole-cell-lysate immunoblotting with specific antibodies was used to monitor the levels of C/EBPβ protein (β-actin was used as control). (b,c) 24 h after transfection, IL-17 (5 ng ml⁻¹) was added to the cells for 2 or 6 h incubation to determine, respectively, Del-1 mRNA expression by qPCR (a) or Del-1 protein levels in culture supernatants by ELISA (c). The qPCR results were normalized to those of GAPDH mRNA and expressed as fold change in transcript levels relative to those of untreated control, the average value of which was taken as 1. (d,e). In a similar experimental setup as in (b,c) HUVEC were treated as indicated and processed for determination of Del-1 mRNA expression by qPCR (d) or Del-1 protein levels in culture supernatants by ELISA (e). (f) ChIP analysis of C/EBPβ binding to EDIL3 promoter in HUVEC which were treated or not with IL-17 (10 ng ml⁻¹; 4 h) with or without 1-h pre-treatment with SB216763 (10 μM; GSK-3β inhibitor), or were transfected with GSK-3β expression vector or empty control vector. IP, immunoprecipitation. (g) HUVEC were treated or not with IL-17 (10 ng ml⁻¹; 30 min), in the presence of SB216763 (10 μM; GSK3β inhibitor) which was added 1 h earlier than IL-17. Cell lysates immunoprecipitated using anti-C/EBPβ were immunoblotted with anti-phospho-C/EBPβ (Thr-188), anti-phospho-threonine antibody, or with anti-C/EBPβ (control). (h) HEK-293T cells were transfected with a hEDIL3-promoter-Luc reporter plasmid along with siRNA to C/EBP (or control siRNA), or with a C/EBPβ expression vector (or control vector) and analysed for luciferase activity after 16 h (left panel). HEK-293T-IL-17RA/C cells were transfected with hEDIL3-promoter-Luc reporter plasmid and C/EBPβ expression vector (or control vector) in the absence or presence of IL-17 (10 ng ml⁻¹) and analysed for luciferase activity after 16 h (right panel). Data are means±s.d. (b–e, n=5; h, n=4 sets of HUVEC cultures). *P<0.01 between indicated groups (ANOVA).