Figure 6: Endothelial MAP4K4 promotes EC activation via NF-κB.

HUVECs were treated with scrambled or MAP4K4 siRNA, and cells were stimulated with 1 or 10 ng ml⁻¹ TNF-α for the indicated times. (a) Cells were seeded onto transwell chambers. Confluent cells were treated overnight with 10 ng ml⁻¹ TNF-α or left untreated, and FITC-labelled dextran that migrated through the HUVEC monolayer was measured. The data represent the mean fluorescence intensity±s.e.m. (analysis of variance (ANOVA) *P<0.05, N=4). (b) THP-1 monocytes were stained with calcein green and adhered to activated endothelium for 30 min. Fluorescence microscopy was performed to determine the number of adherent THP-1 monocytes per microscopic field (× 100). The data represent the mean±s.e.m. as normalized to the unstimulated control time point (ANOVA **P<0.01, ****P<0.0001, N=5). (c–f) Messenger RNA was extracted and quantitative RT–PCR was performed for (c) MAP4K4, (d) ICAM-1, (e) VCAM-1 and (f) SELE. The data represent the mean±s.e.m. as normalized to RPLP0 (*P<0.05, **P<0.005, ^#P<0.0001, N=5–7). (g) Immunoblots were performed for ICAM-1, VCAM-1 and E-selectin. (h) Densitometric analyses from g. The data represent the means±s.e.m. as normalized to VE-cadherin (*P<0.05, N=4–6). (i) Biochemical fractionations were performed, and nuclear fractions were immunoblotted for MAP4K4, p-p65, total p65, p50 and lamin-β1. (j) Densitometric analyses represent the mean±s.e.m. for the 60-min time point as normalized to lamin-β1 (*P<0.05, **P<0.005, N=3). (k) NFκB-luciferase and SV40-Renilla were transfected into HUVECs after treatment with scrambled or MAP4K4 siRNA. Cells were left unstimulated or stimulated with 10 ng ml⁻¹ TNF-α overnight before luciferase and Renilla measurement. The data represent the mean±s.e.m. from four independent experiments. (l–n) HUVECs were transfected with MAP4K4 siRNA and stimulated or not with 1 ng ml⁻¹ TNF-α for 1 h. IgG, p65 and histone antibodies were used to immunoprecipitate chromatin and RT–PCR was used to amplify (l) E-selectin, (m) VCAM-1 and (n) IκBα promoters. The data represent the mean±s.e.m. as normalized to input (*P<0.05, N=3–4).