(A) RPE cells transduced with indicated shRNA constructs were grown on coverslips for 4–5 days to obtain steady state monolayers. Top row, representative immunofluorescence localization of the junctional marker ZO-1. Scale bar = 50 µm. Boxed areas are enlarged to highlight intercellular gaps in Rap1A shRNA cell monolayers (bottom row). Scale bar = 25 µm. Quantification of total monolayer gap area per image field (expressed as % of total field area) using image analysis software (ImageJ) was used as an indication of monolayer integrity. Graph shows the mean of n = 4 fields ± SEM, representative experiment from 2 independent trials. **p<0.01, 1A shRNA compared to both Neg and 1B shRNA. (B) TER was measured on cells prior to shRNA knockdown (day 0), and again after 5 days. Graph shows TER of negative control, Rap1A, or Rap1B shRNA treated cells, representing average TER normalized to control at t = day 0 from n = 4 independent experiments. Control shRNA and Rap1B shRNA cells increased TER after 5 days in culture, while Rap1A shRNA cells did not significantly increase TER from day 0 to day 5. * p≤0.01, NS = not significant; **p≤0.01, TER of Rap1A shRNA monolayers significantly lower at steady state (day 5) compared to control and Rap1B shRNA.