Fig. 6

RIZ2 overexpressing cells release high EGF levels. A EGF concentration in cell culture medium was measured through ELISA method at different time points (24-, 48- and 72 h) in quiescent DLD1-pEGFP and DLD1-pEGFP_hRIZ2. B Immunofluorescence analysis with the anti-EGF antibody (red) on DLD1-pEGFP and DLD1-pEGFP_hRIZ2 positive cells (green). Nuclei are stained in blue. Scale bar, 5 µm. C DLD1-pEGFP and DLD1-pEGFP_hRIZ2 transfected cells were left unchallenged for (24-, 48- and 72 h), avoiding refreshing the medium. EGFR and ERK phosphorylation were revealed by Western blot and densitometry analysis. D DLD1-pEGFP and DLD1-pEGFP_hRIZ2 transfected cells were left untreated for 72 h, avoiding refreshing the medium, in the absence or presence of the anti-EGF neutralizing antibody (anti-EGF nAb). Analysis of EGFR and ERK phosphorylation after treatment with anti-EGF nAb. E DLD1-pEGFP and DLD1-pEGFP_hRIZ2 transfected cells were wounded and left untreated for 30 h avoiding refreshing the medium, in the absence or presence of the anti-EGF antibody (anti-EGF Ab) for analyzing the cell migration induced by conditioned medium. Wound area was calculated using Leica Suite software. Data are presented as the percentage of residual area in wound width over the control cells, analyzed at time 0. Means and SDs are shown. Pictures are representative of three different experiments. ****p < 0.0001; ns not significant