Skip to main content
. Author manuscript; available in PMC: 2015 Aug 10.
Published in final edited form as: Bladder (San Franc). 2015;2(1):e9. doi: 10.14440/bladder.2015.33

Figure 2. Barrier function and tight junction analysis of NHU cultures following claudin 3 shRNA knock-down.

Figure 2

A. Expression of claudin 3, total ZO-1 (both isoforms) and ZO-1α+ proteins was assessed in undifferentiated cultures (U) and in cultures differentiated with 5% ABS and 2 mM calcium (D) by Western blotting, as well as in differentiated NHU cells transduced with control shRNA and CLDN3 shRNA (lanes 3 and 4, respectively). The densitometry analysis of Western blot bands was normalised to the loading control β-actin and the percentage expression relative to control shRNA cells is indicated. B. Compilation of results from shRNA transduction performed on four independent donor NHU cell lines. Expression of CLDN3, CLDN5, ZO-1α− and ZO-1α+ was analysed by western blotting following differentiation of NHU cells transduced with control and CLDN3 shRNA alongside wild-type undifferentiated (U) and differentiated (D) NHU cultures. Expression is plotted as relative expression (± SEM) after normalisation to β-actin. C. Immunofluorescence localisation of claudin 3, ZO-1 (total) and ZO-1α+ in control and CLDN3 shRNA transduced NHU cells following induction of differentiation. Scale bar = 50 μm. D. Barrier function of control shRNA and CLDN3 shRNA transduced cells, alongside undifferentiated (U) and differentiated (D) non-transduced NHU cells, was determined by measurement of transepithelial electrical resistance (TER) after seven days. Results compiled from three independent donor cell lines. Statistical analysis was by ANOVA with Tukey post-test correction. ** P < 0.01; * P < 0.05