Fig. 5

Map1a knockdown (KD) in Sertoli cells by RNAi perturbs the Sertoli cell TJ-permeability function due to changes in the distribution of TJ and basal ES proteins at the BTB. A The regimen used for the studies reported in Figs. 5, 6, and 7. B Following the establishment of a functional Sertoli TJ-barrier (manifested by the presence of stable TER across the cell epithelium cultured on Matrigel-coated bicameral units, see Materials and Methods), Map1a KD was found to perturb the Sertoli cell TJ-barrier after cells were transfected with the Map1a-specific siRNA duplexes versus non-targeting control siRNA duplexes (Table 3). Each data point is a mean ± SD of quadruple bicameral units from a representative experiment, from a total of n = 4 independent experiments using different batches of Sertoli cells, which yielded similar results. *, P < 0.05; **, P < 0.01; ***, P < 0.005, by paired Student’s t-test, compared to the corresponding control. C A study by IB analysis confirmed the KD of Map1a expression without affecting the expression of other TJ- and basal ES-proteins at the Sertoli cell BTB. D Composite data of bar graphs of IB results to illustrate Map1a KD with n = 3 experiments. qPCR results confirmed the KD of Map1a without affecting the expression of Map1b and Map1s using primer pairs specific to the corresponding genes (Table 3). E Results of an in vitro BTB functional assay, illustrating KD of Map1a led to a disruption of the Sertoli BTB, incapable of blocking the entry of a small membrane impermeable biotin (green fluorescence) into the cell cytoplasm, unlike the control Sertoli cells (left panel). KD of Map1a was noted by IF analysis. KD of Map1a led to mis-distribution of TJ (JAM-A and ZO-1) and basal ES (N-cadherin, ß-catenin) proteins, no longer tightly localized at the Sertoli cell–cell interface to support BTB function, instead these proteins were diffusely localized, leading to BTB disruption as noted in (B) and (E, left panel). Composite semi-quantitative data of the top panel are noted in the bar graphs with each bar represents a mean ± SD of n = 3 experiments. At least 50 randomly Sertoli cell pairs were analyzed from each experiment from a total of n = 3 experiments