Effect of Fn52 and Fn52RGDS on fibronectin polymerization and MMP expression in ARPE-19 RPE cells.

<p>Panel A: Immunocytochemistry of ARPE-19 retinal pigment epithelial cells, cultured in the presence of patient-derived vitreous and subretinal fluid, in the presence and absence of scFv antibodies. Staining was done using rabbit anti-fibronectin antibody (Sigma; F3648);FITC-labeled anti-rabbit antibody was used as secondary antibody. Images (60×) were acquired on an Olympus confocal laser scanning microscope. Scale bars represent 10 µm. Panel B: Insoluble fibronectin content in ARPE-19 retinal pigment epithelial cells, cultured as described in Panel A above, was analyzed by deoxycholic acid (DOC) used for the solubilization of Fn multimers, which were generated starting with the same number of cells in each case (control and scFv-treated wells).The blots were developed using anti-fibronectin antibody (1∶5000;Sigma) by chemiluminescence (Amersham Biosciences). The DOC-soluble fraction was analyzed by immunoblotting using mouse anti-GAPDH antibody, as internal control. The figure is a representative image of three separate experiments. Densitometric quantitation of immunoblotting was done, and analysed by the Image-J software; the figure represents mean±SEM (standard error of the mean) calculated from three separate experiments. Lane 1: control - cells in the presence of DMEM and patient-derived vitreous and subretinal fluid (PV); lanes 2–4: cells grown in the presence of PV and 25 µg/ml of O27, Fn52 and Fn52RGDS respectively. Panel C: Zymogram of culture medium supernatants obtained upon culturing ARPE-19 (retinal pigment epithelial cells) cells as described in Panel A, in the absence or presence of scFv antibodies (lane 1: no scFv; lane 2: scFvO27; lane 3: scFv Fn52; lane 4: scFv Fn52RGDS). Densitometric quantitation of the signals in the zymogramwas done, and analysed by the Image-J software; the figure represents mean±SEM (standard error of the mean) calculated from four separate experiments.