Fig. 1

Inhibition of TNFα-induced expression of E-selectin and VCAM-1 by ECPCM. a HAEC were treated with TNFα for 2 h in collection medium or ECPCM, then relative gene expression levels normalized to the collection media control were determined by real-time PCR analysis. Percentage inhibition of gene expression by ECPCM was calculated by comparing treatment with ECPCM and TNFα to treatment with TNFα in collection medium. p-value: ** <0.01 compared to TNFα in collection medium. Data = mean ± SEM. b HAEC were treated for 6 h with or without TNFα in collection medium or ECPCM. Western blot analysis was performed with anti-VCAM-1, anti-E-selectin and anti-β-actin antibodies. The image is representative of three separate experiments. c Relative density quantification of western blot bands for TNFα-treated HAEC in panel (b), performed using ImageJ software. E-selectin and VCAM-1 bands were normalised to the β-actin loading control band. Data = mean ± SEM; n = 3 per treatment. p-value: * <0.05 compared to TNFα in collection medium. d U937 cells were stained with calcein AM and then placed on top of HAEC for 30 min. After three rinses with PBS, U937 attachment to EC was determined by counting the fluorescent cells. Changes in the number of attached cells are expressed as fold changes over the control (treatment in collection medium). p-value: ** <0.01 compared to TNFα in collection medium. Data = Mean ± SEM. e Representative images of U937 cells stained with calcein AM and attached to cytokine-activated HAEC treated in collection medium or ECPCM. Black background = HAEC; white dots = fluorescent U937 cells attached to the HAEC