Figure 1.

Overexpression of plastin 3, which is known to confer organization of actin microfilaments into bundles, perturbs the Sertoli cell TJ-permeability barrier function. (A) Treatment regimen used to transfect Sertoli cells with a full-length plastin 3 cDNA using the mammalian expression vector pCI-neo (pCI-neo/plastin 3) vs. pCI-neo (i.e., empty vector, control) on day 2 for 6 hr, cells were then rinsed twice to remove the transfection reagent. Thereafter, cells were cultured in fresh F12/DMEM until day 5 (i.e., 3 days after transfection for plastin 3 overexpression) before terminated for immunoblotting, or cell staining by fluorescence microscopy using different marker proteins. TER measurement to assess the TJ-barrier integrity was performed daily until day 8. (B) Immunoblotting that confirmed overexpression of plastin 3 in cultured Sertoli cell in vitro had no effects on the expression of selected BTB-associated proteins. (C) Overexpression of plastin 3 (plasmid DNA, both pCI-neo empty vector vs. vector with full-length plastin 3 cDNA construct, pCI-neo/Plastin 3, labeled with Cy3 as described in Materials and Methods appeared as red fluorescence that confirmed successful transfection) was also confirmed by immunofluorescence microscopy, illustrating the fluorescence intensity of plastin 3 (green fluorescence) was induced in pCI-neo/Plastin 3 vs. pCI-neo empty vector which served as a control. Scale bar, 20 µm. Each bar graph in the histograms shown below is a mean ± SD of n = 3 experiments. To assess the intensity of fluorescence signaling of plastin 3, about 25 Sertoli cells were randomly selected from each experiment with n = 3 experiments. (D) Overexpression of plastin 3 was found to perturb the Sertoli cell TJ-permeability barrier. Each data point is a mean ± SD of quadruplicate bicameral units of a representative experiment from n = 3 experiments which yielded similar results. *, P< 0.05.