Figure 6

Overexpression of p-FAK-Y407E phosphomimetic mutant rescues human Sertoli cells from the disruptive effects of PFOS regarding their cytoskeletal and cell adhesion function. (A) Results of a typical TER experiment that monitored the protective effects of p-FAK-Y407E constitutively active phosphomimetic mutant on PFOS (20 µM)-mediated disruption of the human Sertoli cell-TJ permeability barrier function. Cells were transfected with the corresponding plasmid DNA and/or treated with PFOS in specified cell groups on day 3 for 24 hr. Thereafter, TER readings were obtained, and PFOS was then removed by rinsing cells twice with fresh medium (annotated by red arrow), and cells were replenished with fresh medium without PFOS to allow recovery of the TJ-barrier function. TER reading was obtained on a daily basis. Each data point is a mean ± SD of triplicate bicameral units from an experiment. A total of n = 3 independent experiments were performed using different batches of human Sertoli cells which yielded similar results. *P < 0.05; **P < 0.01 by Student’s t-test compared between PFOS-treated and vehicle control groups, or between PFOS-treated vs. PFOS+FAK-Y407E phosphomimetic mutant groups. (B) Regimen used for the experiment summarized in (C) in which human Sertoli cells at ~70–80% confluency were transfected with either empty vector, FAK-WT or FAK-Y407E plasmid DNA, and with or without PFOS for 24 hr. Cells were allowed to recover for 24 hr before termination and used for IF. (C) Immunofluorescence analysis of F-actin (green fluorescence) and β-tubulin (green fluorescence) network as well as microtubule regulatory protein EB1 (green fluorescence) and adhesion protein β-catenin (green fluorescence) in human Sertoli cells. Overexpression of p-FAK-Y407E, but not FAK-WT, in human Sertoli cells together with PFOS treatment was capable of rescuing these cells from the disruptive effects of PFOS regarding the organization of F-actin and MTs in these cells. FAK-Y407E overexpression also promoted proper spatial expression of EB1 across the Sertoli cell and proper localization of ß-catenin at the Sertoli cell-cell interface. Successful transfection was confirmed by red fluorescence wherein plasmid DNA was labeled with Cy3. Cell nuclei were visualized by DAPI. Scale bar, 40 μm, which applies to all other micrographs.