Figure 3.

Insulin induces oBRB breakdown regardless of glucose concentration. (A) Real-time impedance of RPE monolayers treated with 5- or 25-mM glucose ± 100-nM insulin. Endpoint quantification is shown as mean ± SD (n = 3). ****P < 0.0001 (one-way ANOVA with Brown–Forsythe test and Tukey's test). (B) Dose-dependent effect of insulin (0.1–100 nM) on RPE impedance. Quantified endpoints are shown as mean ± SD (n = 3 or 4). *P < 0.05; ****P < 0.0001 (one-way ANOVA with Brown-Forsythe correction and Dunnett's test). (C) Western blot of RPE cells treated with 25-mM glucose ± 100-nM insulin, expressed as mean ± SD (n = 3). *P < 0.05 (unpaired two-tailed t-test). (D) Immunofluorescence for claudin-19 and ZO-1 under the same conditions. Scale bar: 25 µm. (E) Quantification of junctional parameters using Junction Mapper, expressed as mean ± SD (n = 1099). ****P < 0.0001 (unpaired t-test). (F) RT-qPCR of claudin transcripts normalized to GAPDH and inter-claudin expression, reported as mean ± SD (n = 3). (two-way ANOVA with Sidak's correction); ns, not significant. HG, high glucose; HGI, high glucose insulin.