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. 2015 Jul 22;6(29):28389–28400. doi: 10.18632/oncotarget.4949

Figure 3. Effect of trypsinogen 4 silencing on the migration ability of tumor endothelial cells.

Figure 3

Migration was assessed in a wound healing assay; wound closure was monitored by Cell®Imaging Station and quantified by measuring the wound area over time and comparing it with the initial size (assumed as 100%). Results are the mean of three replicates. Representative captions of the wounds are shown. Tumor-EC from ovarian carcinoma (HOC-EC) and normal-EC from derma and lung (D- and L-HMVEC) were transfected with either trypsinogen 4 (Tryp4) or not-targeting (NT) siRNA. Silencing was assessed by RT-qPCR 24h later (i.e. at the time of wounding); trypsinogen 4 expression before transfection was arbitrarily taken as the reference. Different populations of HOC-EC were investigated (populations 13, 15, 17 and 18); shown here are representative results. A: The angiogenic milieu triggers the migration of tumor-EC. Shown here are the results of HOC-EC embedded (+, black) or not (−, white) in the in vitro reconstituted “angiogenic milieu” (i.e. human recombinant VEGF-A, FGF-2 and EGF). B: Trypsinogen 4 silenced tumor-EC are not longer able to close the wound.Shown here are the results of HOC-EC, silenced (Tryp4 siRNA) or not (NTsiRNA and wild type), assayed in presence of the angiogenic milieu. C: Trypsinogen 4 does not have an impact on the migration of EC from normal tissues. Shownhere are the results of HOC-EC, D-HMVEC and L-HMVEC silenced (Tryp4 siRNA) or not (NT siRNA) assayed in presence of the angiogenic milieu. D: Tumor-EC deprived of the angiogenic milieu migrates weakly regardless of trypsinogen 4. Shownhere are the results of HOC-EC silenced (Tryp4 siRNA) or not (NT siRNA and wild type) assayed in absence of the angiogenic milieu.