Fig. 3

The validation of lentiviral vector-mediated overexpression of RFC1 protein in vivo, which led to an increase in the proteins of BRB. A The Western Blotting image shows corresponding overexpression of RFC1 protein in RFC1-LV transduced neuroblastoma cells (N2a) compared to Control-LV transduced cells. For cell culture lysates, Beta-Actin was the loading control. The graph illustrates the quantification of the relative band densities of given Western blotting image which expresses RFC1 band density in proportion to β-Actin, which shows increase in relative protein levels in RFC1-LV treated retinas (*p = 0.016). B The confocal images from whole mount retinas confirmed the intravitreal delivery of Control-LV into the mouse eye showing lentiviral vectors infected various retinal cells, as reporter gene GFP (green) signal indicated. Also, as magnified image showed, perivascular cells and pericytes expressed GFP protein in LV-RFC1 or Control-LV injected retinas, microvessel trace was shown by dashed line. C The representative images of whole mount retinas which had been treated by LV-RFC1 showed increased RFC1 immunosignal (red) compared to Control-LV injected control retinas (n = 3). D The graph illustrates the percentage of mean grey value of RFC1 frame in lectin positive microvessel area in RFC1-LV treated group normalized to Control-LV treated group as described in Methods section. RFC1-LV delivery significantly increased the percent of the mean grey values of RFC1 (n = 3). E–H RFC1-LV delivery significantly increased the percent of the mean grey values of collagen-4 (n = 4), occludin (n = 3), claudin-5 (n = 3), but not ZO-1 compared to Control-LV delivered groups. (*p ≤ 0.05). Collagen-4 (magenta), occludin (cyan) lectin (Yellow) immunosignal increased as well as claudin-5 (magenta) except ZO-1 (cyan) via RFC1-LV treatment compared to Control-LV treatment. Nuclei were labelled with Hoechst 33258 (blue) Data are mean ± S.E.M. Mann-Whitney U; Scale bars= 10 μm