Growth factors induced cell migration of ARPE19 and RPE50.

<p>(A) ARPE 19 was cultivated with a wound healing culture insert and serum-starved for 48 h. After removal of culture insert (0 h), cells were treated with 50 nM of HGF (H50), EGF (E50), TGFβ1 (T50) or PDGF (P50), 50 ng/ml HB-EGF (HB-E50), 25 nM HGF coupled with 25 nM EGF (H25/E25) or 25 nM HGF coupled with 25 ng/ml EGF (H25/HB-E25) in serum free medium for 18 h and photographed. The cells migrated into the blanking area between the indicated orange lines representing the boundary between the blanking area and cell culture at 0 h. The results were representatives of four reproducible experiments. (B) Quantitative analysis of relative migration of ARPE19 (gray) and RPE 50 (black) treated with growth factors as indicated in (A) and supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044937#pone-0044937-g001" target="_blank"><b>Figure 1</b></a>, respectively. The number of cells migrated into the wound area were counted under phase contrast microscope (at 200-fold magnification). For each treatment, total cell numbers in four microscopic fields were counted. Relative migration of the cells were calculated as the ratio of cell numbers of each treatment <i>vs.</i> that of untreated cells, taken the ratio of untreated cells as 1.0. The results were averages of three experiments with coefficient of variation (C.V.) of 5.5%. (*) and (**) represent statistical significance (P<0.05 and P<0.005, respectively) for comparison of the growth factor-treated with untreated group. (^##) represent statistical significance (P<0.005) for comparison of the combined growth factors <i>vs.</i> single growth factors group as described in the text. (C) and (D) RPE 50 (black) and ARPE19 (gray) were untreated (-), treated with 50 nM HGF (C) and EGF (D), or HGF and EGF coupled with DMSO (as solvent control) or various inhibitors as indicated. Wound healing assay were performed and relative migrations were quantitated as described in (B). JNJ in (C): JNJ38877605. The results were averages of 3 experiments with coefficient of variation (C.V.) of 5.5%. (**) represent statistical significance (P<0.005) for comparison of the inhibitor- <i>vs.</i> DMSO-treated group.