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. 2016 Jun 27;7(30):48481–48500. doi: 10.18632/oncotarget.10311

Figure 6. Treatment of TNF-HUVEC with soluble recombinant human ephrinA4 strongly potentiates survival of CLL cells and a possible transcellular TEM pathway.

Figure 6

A. Soluble ephrinA4 levels in the serum of CLL patients were determined by indirect ELISA (ng/mL) and possible associations with clinical parameters examined. B. CLL cells from 12 patients having (LApos) or not (LAneg) lymphadenopathy, used in the EphA2Fc or siRNA assays (Figures 3 and 4), were left transmigrating 12 hours through TNF-α preactivated HUVEC monolayers preincubated (30 min) with saturating amounts of recombinant human ephrinA4 (HUVEC+ephrinA4Fc), purified human Fc (HUVEC + hFc) or untreated before addition of CLL cells. i) Absolute number of transmigrated cells (TM) was counted by flow cytometry and expressed as percentage of total input cells (TEM rate). ii) Absolute levels of viable CLL cells in suspension cultures or in TEM assays (non-TM and TM cells) in the ephrinA4Fc or hFc treated TEM assays were compared to untreated conditions. iii) Relative viability of TM cells normalized to basal levels (suspension cultures) or to the corresponding non-TM cells in the ephrinA4Fc or hFc treatments compared to untreated TEM assays. C. i) Transwell filters were stained with anti VE-Cadherin (green, HUVEC junctions) and Hoechst (blue, nuclei) and examined by confocal microscopy. Adhered CLL cells were located at VE-Cadherin interendothelial junctions or separated from them. ii-iii) Caveolin-1 and ICAM-1 immunofluorescence staining of TEM assays done in chamber slides 2 hours after coculture with LApos CLL cells. ii) CLL cells inside of HUVEC were surrounded by caveolin-1 endothelial caveola (asterisks, > 1μm from VE-Cadherin junction) and ICAM-1. Insets show Z- views of caveolin and ICAM-1 staining for a a transcellular (TC) and a paracellular (PC) TEM CLL nuclei. iii) Measurement of CLL cells located within caveolin-1, at VE-Cadherin junctions or unclassified in ephrinA4Fc treated compared to untreated TNF-HUVEC after 2 hours coculture (at least 200 nuclei were counted per condition). Paired two-tailed Student's T test significance values: * P<0.05; ** P<0.01; *** P<0.001.