Fig. 2

The validation of Accell siRNA mediated in vivo RFC1 knockdown, which led to a reduction in critical proteins of the inner BRB. A Western blotting image of retinas (n = 4 mice/group) depicting the robust decrease in RFC1 protein of RFC1-siRNA delivered ones compared to scrambled-siRNA. β-Tubulin III was loading control. 35 µg protein was loaded for each retina. B The graph illustrates the quantification of the relative band densities of given Western blotting image which expresses RFC1 band density in proportion to β-Tubulin III, which shows decrease in relative protein levels in RFC1-siRNA treated retinas (*p = 0.028). C The graph shows that RFC1-siRNA delivery reduced retinal RFC1 mRNA levels by 24.75% when compared to scrambled-siRNA (*p = 0.004). D The graph illustrates the percentage of mean grey value of RFC1 frame in lectin positive microvessel area in RFC1-siRNA treated group normalized to scrambled-siRNA treated group as described in Methods section. RFC1-siRNA delivery significantly reduced the percent of the mean grey values of RFC1 by 72.10% (n = 4). Representative confocal images of Scrambled-siRNA or RFC1-siRNA delivered retinal microvessels stained by anti-RFC1 antibody (red). E RFC1-siRNA treated retinal microvessels showed less NG2 immunosignal compared to scrambled-siRNA treated ones, indicating that pericytes might also be damaged by RFC1-siRNA. F However, juxtanuclear diameter ratio of total of 97 pericyte-associated microvessels were measured (n=3/group) and no significant changes were found. The dots represent the mean of each juxtanuclear section per pericyte and divided by the initial diameter of each vascular segment. G Also, pericyte body counts per microvessel length (mm) were not different between the groups, n = 3 images were analyzed per animal. H Whole mount retinas were labeled by anti-RFC1 antibody (red) and Fluorescein lectin (green). RFC1 immunosignal was decreased and interrupted along deep retinal microvessels. Also, lectin signal was weakened in RFC1-siRNA treated retinas representing the structural decomposition of microvessels. I-L RFC1-siRNA delivery significantly reduced the percent of the mean grey values of collagen-4 68% (n=4), occludin 57.03% (n=3), claudin-5 76.26% (n=4), and ZO-1 53.22% (n=4). The reductions that are statistically significant represented as *p ≤ 0.05. Nuclei are labelled with Hoechst 33258 (blue), Data are mean ± S.E.M, Mann–Whitney U; Scale bars= 10 μm