Fig 2: Validation of adhesion complex-localized phosphorylation sites.(a) Immunoblot analyses of the protein complexes isolated from cells spread on FN or Tf for 120 min. Samples were probed using phosphospecific antibodies targeting: paxillin pTyr118; FAK pTyr397, pTyr576 and pTyr407; MYPT1 pThr696; cortactin pTyr421; SHP2 pTyr580; and PKCd pTyr313. (b) Immunofluorescence images of cells spread on FN for 120 min. Cells were stained using the phosphospecific antibodies FAK pTyr407, cortactin pTyr421 and PKCd pTyr313 (all green). In each case, cells were also stained using an antibody targeting the focal adhesion marker paxillin (red). Scale bar, 20 µm. (c) Immunoblot analyses of the WCLs of cells spread on FN or Tf for 120 min. Samples were probed using the same antibodies as used to probe isolated complexes in a. Dashed lines in a,c indicate where the images have been cropped for display purposes. White arrows to left of blots highlight the expected MWs of the corresponding proteins. Black arrows to the left of blots highlight a putative high MW isoform of PKCd.

Fig 3: SC79 blocks PFOS-induced disorganization of actin microfilaments by maintaining proper spatial expression of actin regulatory proteins in Sertoli cells exposed to PFOS. Sertoli cells cultured at 0.03 × 106 cells/cm2 for 3 days were pre-treated with 2 µg/ml SC79 (5.5 µM) for 30 min, and then cells were rinsed and treated with 20 µM PFOS for 24 hr. Cells were then fixed and visualized by immunofluorescence microscopy. It was noted that Sertoli cells exposed to PFOS without pre-treatment with SC79 were grossly disorganized in which actin microfilaments were truncated, likely the result of changes in the spatial expression of Arp3 (a branched actin polymerization protein), palladin (an actin cross-linking and bundling protein) and p-FAK-Y407 (a BTB-associated signaling protein known to promote BTB integrity14). However, pretreatment of Sertoli cells with SC79 blocked the PFOS-induced disorganization of actin microfilaments, causing actin microfilaments to be organized similar to control cells even in the presence of PFOS for 24 hr. This blocking effect of SC79 on PFOS-induced Sertoli cell injury apparently was mediated by maintaining the proper spatial expression of Arp3, palladin and p-FAK-Y407 as noted in control cells. Distribution of the target proteins Arp3 and p-FAK-Y407 at the cell cortical zone was also quantified by measuring the fluorescence intensity at the Sertoli cell-cell interface (see dotted white rectangles in control cells vs. dotted yellow rectangles in PFOS-treated cells), which was the average of 4 measurements (i.e., fluorescence intensity) at two opposite ends of a Sertoli cell as annotated by the rectangles for each cell. About 70 randomly selected cells in each experiment were quantified with n = 3 independent experiments. Scale bar, 30 µm, which applies to all other micrographs.

Fig 4: PFOS perturbs Sertoli cell BTB function through changes in the expression and/or localization of proteins at the immunological barrier. Sertoli cells cultured on Matrigel-coated bicameral units, dishes or coverslips were treated on day 3 with 10, 20 or 50 µM PFOS for 24 hr. Thereafter, cells were washed twice with PBS to remove residual PFOS and terminated for immunoblotting (IB) or IF on day 4. (A) Typical results of a TER experiment that monitored the effects of PFOS, at 20 µM and 50 µM vs. vehicle control (DMSO) on the Sertoli cell TJ-permeability barrier. Each data point is a mean ± SD of triplicate units from an experiment, which was repeated 3 times and yielded similar results. **P < 0.01 by Student’s t-test compared between PFOS-treated and control cells. (B) Immunoblot analysis to assess the effects of PFOS on the expression of TJ proteins: occludin, CAR and ZO-1; basal ES proteins: N-cadherin and ß-catenin; actin regulatory proteins: Arp3, Eps8, palladin, formin 1 and plastin 3; signaling molecules: activated Akts: p-Akt1-T308, p-Akt1-S473, pAkt2-S474 vs. Akts (including Akt1, 2 and 3), and activated FAK: p-FAK-Tyr407 vs. FAK; MT regulatory proteins: EB1 and detyrosinated a-tubulin. Actin and a-tubulin served as protein loading controls. PFOS was found to down-regulate p-Akt1-T308, p-Akt1-S473, p-Akt2-S474 and p-FAK-Tyr407. Composite data of immunoblottings are shown in Figure S1A; and uncropped blots are shown in Figure S4. (C) Immunofluorescence analysis to assess the effects of PFOS at 20 µM and 50 µM vs. control on the distribution of TJ proteins: occludin, CAR and ZO-1; basal ES proteins: N-cadherin and ß-catenin. PFOS caused internalization of TJ and basal ES proteins in Sertoli cells in which these proteins no longer tightly localized at the Sertoli cell-cell interface. Occludin and CAR at the cell-cell interface (annotated by white arrowheads) were diminished following PFOS treatment (yellow arrowheads). For ZO-1, N-cadherin and ß-catenin, these proteins were localized tightly at the cell-cell interface (white brackets) but were considerably internalized, diffusing away from the cell cortical zone (yellow brackets). Sertoli cell nuclei were visualized by DAPI. Composite analyzed data are shown in Figure S1B. Scale bar, 30 µm.

Fig 5: Immunofluorescence analysis to assess the effects of PFOS on the organization of actin microfilaments in Sertoli cells via changes on the distribution of actin regulatory proteins. Sertoli cells cultured for 3 days were treated with 20 µM PFOS for 24 hr. Cells were then harvested and used for IF, and results from two different experiments were shown in middle and right columns vs. the control cells in the left column. PFOS caused truncation and defragmentation of F-actin network (green and gray). These changes in the organization of actin microfilaments were likely the result of changes in the spatial localization of several actin regulatory proteins. Arp3, a branched actin polymerization protein that effectively converted actin microfilaments into a branched/unbundled configuration no longer localized at the cell-cell interface to support F-actin dynamics at the cell cortical zone. Instead, Arp3 was internalized and found in the Sertoli cell cytosol, thereby impeding actin microfilaments to organize as long actin bundles stretched across the cell cytosol. Palladin, an actin cross-linking and bundling protein, no longer stretched across the Sertoli cell cytosol to maintain actin microfilaments across the Sertoli cells as found in control cells. Instead, palladin was retracted from the cell peripheries but concentrated to cell nuclei after PFOS treatment. p-FAK-Y407, a signaling protein shown to be involved in maintaining BTB integrity by maintaining F-actin network in Sertoli cells as actin filament bundles also no longer localized to the Sertoli cell-cell interface to maintain the TJ-barrier function. Instead, it was internalized into the cell cytosol, thereby destabilizing the TJ-barrier function. Sertoli cell nuclei were visualized by DAPI. Scale bar, 30 µm.