Fig 2: Immunofluorescence staining against claudin-11 reveals a structural disturbance of the BTB (a,b) in gcPex13?ex2/?ex2/Stra8-Cre+/- testes that was functionally assayed by Evans Blue injection (c,d). (a) Claudin-11 (red) was concentrated in the suprabasal layer within the germinal epithelium of control testis. (b) In the gcPex13?ex2/?ex2/Stra8-Cre+/- mice, the immunoreactivity was also extended focally up to the lumen of the seminiferous tubules. (c) In control, the azo-dye Evans Blue was clearly located in the suprabasal epithelium and in the interstitium. (d) In gcPex13?ex2/?ex2/Stra8-Cre+/- testes, Evans Blue was also dispersed adluminal of the germinal epithelium. Evans Blue is indicated in red. Nuclei DAPI stains are shown in grey. Bars = 50 µm.

Fig 3: Nf1 Loss or HRas Activation in Oligodendrocytes Causes Optic-Nerve Enlargement, Myelin Decompaction, Loss of Claudin-11, and Altered Cx32 Localization(A) Gross micrographs of optic nerves at the level of the chiasm in 12-month-old animals. Ruler shows 1 mm markings.(B) Toluidine-blue-stained semithin optic-nerve cross-sections, 1 mm rostral to the chiasm.(C) Optic-nerve g-ratio scatterplots (~5,000 axons/graph; measurements 1 mm rostral to the chiasm in three to five electron micrographs at 10,000×). Red box: 99% of WT g-ratios superimposed upon graphs of other genotypes. ANOVA, Tukey post hoc, p = 2.2 × 10-16.(D) Diagram of myelin compaction. Brown circle: axon cross-section; gray: major dense lines (MDLs); orange: Cx32 GJs between compact myelin layers; blue lines: claudin-11+ TJs in compact myelin.(E) Electron micrographs (scale bar, 10 nm) of WT, PLPCre;Nf1 fl/fl, and CNP-HRas optic-nerve myelin. White arrows: intraperiod lines (ILs); black arrows: MDLs.(F–I) Western blots of total optic-nerve lysate or whole-brain cytosolic, endosomal, or myelin fractions showing myelin proteins (F), claudin-11 (G), or Cx32 cellular localization (H and I). n = 3–5 animals/genotype/experiment.See also Figures S1, S2, S3, and S4.

Fig 4: GO enrichment analysis. (A) The enriched GO biological processes. The GO annotations were divided into two groups namely activated and suppressed functions by R package clusterProfiler with gseGO function. The x-axis represents the gene ratio for each GO term. The circle size reflects the number of genes. (B) GSEA-based GO enrichment plots by R package enrichplot. Green line indicates the running enrichment scores. The bar color represents the activated (red) or suppressed (blue) functions. (C) The connection of genes among the prominent GO functions by enrichGO function. The circle color represents the different regulations of genes with log FC values (red; upregulation, green; downregulation). The arrow indicates CLDN11 gene.

Fig 5: Disruptive effects of PFOS on Sertoli cell F-actin organization and distribution of BTB-associated proteins at the cell-cell interface.(A) Treatment regimen used for this experiment. Sertoli cells cultured alone at 0.4 × 106 cell/cm2 for 3 days following the establishment of a functional TJ-barrier were treated with 20 µM PFOS for 24 hr. Thereafter, cells were harvested for immunoblotting or for immunofluorescent microscopy. (B) Immunoblot analysis to assess changes in the steady-state level of Cx43 (a GJ protein), TJ- (e.g., claudin 11), basal ES (e.g., N-cadherin, ß-catenin), and actin binding/regulatory proteins (e.g., Arp3, N-WASP, formin 1 and Eps8) with ß-actin serving as the protein loading control. Cx43 appeared as three immunoreactive bands designated as p0, p1 and p2, corresponding to Mr of 39, 41, and 43 kDa due to differential phosphorylation, which is consistent with earlier reports5657. A study by qPCR (with GAPDH serving as the internal control for normalization) also confirmed a down-regulation of Cx43 expression. Immunoblot analysis also illustrated a down-regulation on the steady-state level of Cx43 protein when p0, p1 and p2 isoforms were analyzed as a whole, with a more considerable reduction for p2. Besides Cx43, claudin 11 and ß1-integrin were also down-regulated. Each bar in the two bar graphs is a mean ± SD of n = 5 experiments. **P < 0.01. (C) F-actin organization in Sertoli cells was assessed by phalloidin-FITC staining, illustrating PFOS induced truncation and defragmentation of actin microfilaments. PFOS treatment also considerably down-regulated the expression of Cx43 and claudin 11 at the Sertoli-Sertoli cell interface, consistent with data shown in (B). While the levels of N-cadherin and ß-catenin were not considerably affected based on immunoblotting results, these basal ES proteins localized diffusely at the Sertoli cell-cell interface, apparently rapidly internalized. Different cell densities were used to optimize the visualization of different target proteins including F-actin as noted in Materials and Methods. Micrographs shown herein are representative results of an experiment. All three independent experiments yielded similar results. Scale bar, 25 µm, which applies to all other micrographs. Each bar graph is a mean ± SD of n = 3 experiments. **P < 0.01.