Fig. 6

Characterisation of EA.hy cell lines and the influence of the EPLIN isoforms on transendothelial migration of neutrophils. (A) Transendothelial resistance of EA.hy WT and KO cells during cell growth. (B) Western blot analysis of EA.hy cell lines before or after re-expression of each of the EPLIN isoforms and subsequent activation by 50 ng/ml TNF-α. GAPDH served as an internal control. Full immunoblots are shown in supplementary Fig. S6 (C) The indicated EA.hy cell lines were cultured on trans-well filter membranes. TEM of neutrophils was determined by counting of transmigrated neurophils from the lower compartment. TEM is reduced in Eplin KO cells (p = 0.0425), while re-expression of EPLIN-α-EGFP (EPL-α-EGFP cells) returned TEM to control levels, seen in WT EA.hy cells (p = 0.7893). In contrast the re-expression of EPLIN-β-EGFP (EPL-β-EGFP) showed only a moderate increase in TEM but still remained significantly lower compared to WT EA.hy cells (p = 0.0306). Data were analyzed from 4 independent experiments. Error bars represent ± SEM, *, P < 0,05. **, P < 0,005. ns = not significant. Kruskal–Wallis test was done for the multi comparison of different groups. (D) EA.hy cell lines as indicated were analysed by LSM. Actin filaments were labelled with phalloidin-TRITC (white). EA.hy WT and EA.hy KO cells did not express fluorescently-labelled proteins and were therefore immunolabelled indirectly using anti-pan-EPLIN (green channel). Re-expression of EPLIN-α-EGFP and EPLIN-β-EGFP in EA.hy cells as indicated (cropped accordingly). EPLIN labelling has also been cropped as indicated. Actin filaments (in black) and EPLIN labelling (in green) are shown at the corresponding positions in the line scans. Images shown are representative of three independent experiments. Scale bar, 10 µm. (E–J) FRAP analysis of EPL-α-EGFP, F and EPL-β-EGFP locolized near junctional actin filaments (JA) in the EA.hy KO cells. (E) Representative images of a FRAP experiment in EA.hy KO cells expressing EPL-α-EGFP (upper panel) or EPL-β-EGFP (lower panel). Scale bar, 1 μm. (F) Mean values of the normalized fluorescence recovery intensities ± SEM of EPL-α-EGFP (n = 27 areas) and EPL-β-EGFP (n = 31 areas), from 4 independent experiments. (G and H) Quantification of mobile fraction and half life of the EPL-α-EGFP or EPL-β-EGFP (p = 0.0020). (I and J) Quantification of mobile fraction and half life of the EPL-β-EGFP localized at SFs, upon TNFα stimulation. (unpaired t-test, p = 0.0087).