Fig. 2

HECTD3 promotes adhesion of tumor cells to HUVECs by upregulating E-selectin, ICAM-1 and VCAM-1 expression in HUVECs. a RNA-sequencing analysis of gene expression in untreated and TNFα-treated control and HECTD3 knockdown HUVECs for 2 h. Heat map showing the expression of genes responsive to TNFα in untreated control HUVECs (siCo_0h), TNFα-treated control HUVECs (siCo_2h), untreated HECTD3 KD HUVECs (siH_0h) and TNFα-treated HECTD3 KD HUVECs (siH_2h). Each group has two experimental repeats. b Immunoblot analysis of adhesion molecules, like E-selectin, ICAM-1 and VCAM-1 in HUVECs knocking down HECTD3 or p65 using corresponding siRNA for 36 h, and stimulated with or without LPS (300 ng/mL) as indicated time. siControl (siCtrl) targeted nothing and siHECTD3 (siH) was a siRNA pool containing siHECTD3 1# and 2# here and in the following experiments. NC, negative control. NS, nonspecific band. c qRT-PCR analysis of adhesion molecules in HUVECs knocking down HECTD3 or p65 and stimulated with LPS (300 ng/mL) for 2 h. d Schematic representation of the in vitro adhesion assay. HECTD3 or p65 was knocked down in HUVECs, and cells were seeded into 6-well plates. HUVECs were treated with LPS (300 ng/ml) or TNFα for 4 h when the cells became fully confluent. Then, suspended GFP-labeled tumor cells were added and incubated for 1 h. Unattached cancer cells were washed away, and cancer cells adhered to HUVECs were quantified. e Representative images of the adhesion of GFP-labeled tumor cells to monolayer-cultured HUVECs transfected with the indicated siRNA and stimulated with or without LPS. f Bar graphs show the number of GFP-labeled tumor cells attached to monolayer-cultured HUVECs of panel e. g HUVECs stably overexpressing siRNA-resistant HECTD3, HECTD3 C823A mutant, and control (pCDH) were established. Immunoblots of these HUVECs knocked down endogenous HECTD3 or not and stimulated with LPS (300 ng/ml) for 4 h. h HUVECs stably overexpressing siRNA-resistant HECTD3, HECTD3 C823A mutant, and control (pCDH) were established. qRT-PCR analysis of these HUVECs knocked down endogenous HECTD3 or not and stimulated with LPS (300 ng/ml) for 2 h. i Representative images of the adhesion of GFP-labeled tumor cells to monolayer-cultured HUVECs in which HECTD3 was overexpressed or not. E-selectin, ICAM-1 and VCAM-1 were simultaneously knocked down by siE+I + V, a siRNA mixture of siE-selectin, siICAM-1 and siVCAM-1. j Quantitative data of panel i. Data represent 3 independent experiments for all of the above experiments. Data are presented as the mean ± SEM, and statistics were calculated using a two-tailed t-test for c, f, h, j. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant. Scale bars, 200 μm for e, i