Supplementary Figure 7: Experimental timeline for virally mediated HIPP injections and rescue experiment, virus validation and behaviors

Experimental timeline (A) and virus validation (qPCRs, **p = 0.0039, Western Blots, ***p = 0.0006) (B) for AAV-shRNA and AAV-shRNA-cldn5 injections in the HIPP. Full-length blots are included in Supplementary Fig. 12. Downregulation of cldn5 expression had no effect in the splash (C) and sucrose preference tests (D). However mice injected with AAV-shRNA-cldn5 spent more time immobile in the forced swim test (E) and stressed AAV-shRNA-cldn5 mice displayed social avoidance (F). Representative heat maps are shown on the right. G) Experimental timeline of the control cohort for rescue experiment. H) Removal of Dox from the water allowed recovery of cldn5 expression at both mRNA (p = 0.9473) and protein (p = 0.2077) levels in the rescue cohort. Full-length blots are included in Supplementary Fig. 12. I) Conversely, downregulation of cldn5 mRNA (**p = 0.0020) and protein (*p = 0.0116) levels was confirmed in the cohort remaining on Dox. Full-length blots are included in Supplementary Fig. 12. G) Stressed AAV-shRNA-cldn5 mice that remained on dox treatment displayed depression-like behaviors in forced swim (**p = 0.0015) and sucrose preference (**p = 0.0028) tests before (J) and after 5-day rest as measured with sucrose preference (***p = 0.0002) and social interaction tests (**p = 0.0028) (K). Representative heat maps are shown on the left. Data represent mean ± SEM, number of animals (n) is indicated on graphs. Two-way ANOVA followed by Bonferroni’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001