Fig. 3

HECTD3 increases the expression of adhesion molecules by stabilizing IKKα and recruiting nuclear IKKα to adhesion molecule gene promoters. a Immunoblot analysis of the expression of IKKα and activation degree of the NF-κB signal pathway. HUVECs knocking down HECTD3 were stimulated with LPS (300 ng/ml) for indicated time. b A comparison of the expression of the adhesion molecules in HUVEC knocking down HECTD3, IKKα and p65, and stimulated with or without LPS (300 ng/mL) as indicated time. c qRT-PCR analysis of adhesion molecules in HUVECs knocking down IKKα and stimulated with or without LPS (300 ng/mL) for 2 h. d IKKα overexpression largely rescued the HECTD3 KD caused reduction of E-selectin and ICAM-1, and partially rescue the reduction of VCAM-1 in HUVECs. HUVECs were treated with TNFα (10 ng/ml) for indicated time and different proteins were detected by WB. e IKKα overexpression largely rescued HECTD3 KD caused reduction of adhesion phenotype. Representative images of the adhesion of GFP-labeled tumor cells to monolayer-cultured HUVECs are shown. Scale bar, 200 μm. f Bar graphs show the number of GFP-labeled tumor cells attached to monolayer-cultured HUVECs from panel e. g IKKα and IKKβ was transiently knocked down in HECTD3-overexpressing HUVECs stimulated with LPS (300 ng/ml) and HECTD3 overexpression-induced increases of adhesion molecule expression were blocked when IKKα or IKKβ was depleted. h HECTD3 positively regulated IKKα and H3S10ph levels in HUVECs. Left: HECTD3 was knocked. Right: HECTD3 was stably overexpressed in HUVECs. i Chromatin immunoprecipitation (ChIP) assays were performed using an anti-IKKα antibody in HUVECs transfected with siControl or siHECTD3 and stimulated with LPS (300 ng/mL) for 1 h. j qPCR results of the samples in panel i. k HECTD3 knockdown by siRNA decreased IKKα protein stability in HUVECs. The cells were incubated with 50 μg/ml CHX for the indicated times and were collected for immunoblotting. Tubulin was used as the internal control. The band intensity of IKKα at each time point was quantified using ImageJ. The experiments were repeated three times, and a representative experimental result is presented. l Quantitative data of panel k. m HECTD3 knockdown induced IKKα protein degradation through lysosomes but not proteasomes. HUVECs were treated with lysosome inhibitors (NH₄Cl, 10 mM and HCQS, 50 μM, overnight) or proteasome inhibitor (MG132, 20 μM for 6 h) after knocking down HECTD3. Data represent three independent experiments for all of the above experiments. Data are presented as the mean ± SEM, and statistics were calculated using two-tailed t-test for c, f, i, two-way ANOVA for l. *P < 0.05; **P < 0.01; ***P < 0.001