Figure 3

Recombinant CCN5 protein inhibits EMT induced by aflibercept (Afl) in mouse eyes. (A) An experimental scheme. Normal human IgG (40 mg/eye), Afl (40 μg/eye), and Afl and CCN5 (40 ng/eye) were intravitreally injected. Mice were sacrificed, and eyes were enucleated at 14 days post-injection. (B) Flat mounts of RPE/choroid complex were stained with anti-α-SMA antibody. Representative images are shown. Scale bar, 1000, 500, and 250 μm. (C) The intensity of α-SMA positive areas was quantified by Image-J software and plotted. (D) Flat mounts of RPE/choroid complex were stained with anti-α-SMA and anti-ZO-1 antibodies, and Hoechst 33342. Representative images are shown. Scale bar, 50 μm. (E) The mean number of RPE cells in 100 × 100 μm area was manually counted and plotted. (F) The mean size of RPE cells in 100 × 100 μm area was manually calculated and plotted. Error bars = SEM. (n = 8, Cont; n = 8, IgG; n = 10, Afl; n = 13, Afl+CCN5; one-way ANOVA; ***p < 0.001).