Figure 1. A study to assess whether formin 1 structurally interacts with MTs in Sertoli cells in the seminiferous epithelium of rat testes.

A, Dual-labeled IF analysis was performed to assess whether formin 1 colocalized with β-tubulin, the building block of MTs, because MT is a polymer of α-/β-tubulin dimers, in Sertoli cells cultured in vitro with an established TJ-permeability barrier. Indeed, formin 1 (green fluorescence) colocalized with β-tubulin (red fluorescence, ie, MTs) and also F-actin (red fluorescence) in Sertoli cell cytosol. Cell nuclei were visualized by DAPI (blue). Scale bar, 20 μm, which applies to other micrographs in the same panel. B, Localization of formin 1 in the seminiferous epithelium of adult rat testes. PFA-fixed paraffin cross-sections of adult rat testes stained with formin 1 (green fluorescence), illustrating its localization at the apical ES and basal ES/BTB and also the stalk-like structures across the epithelium in tubules of stages VI–VII (lower panel). Formin 1 (green fluorescence) was not found in control sections (upper panel) incubated with normal mouse IgG, which substituted the primary antibody to be followed by an incubation with the corresponding goat antimouse IgG-Alexa Fluor 488 secondary antibody (Table 1), illustrating staining shown in the lower panel was specific for formin 1. Scale bar, 150 μm, which applies to the other micrograph. C, Stage-specific expression of formin 1 (green fluorescence) in the seminiferous epithelium of adult rat testes using PFA-fixed paraffin cross-sections was noted in which formin 1 staining was limited to stage VI-VII tubules, colocalized with β-tubulin (red fluorescence) and EB1 (red fluorescence) in particular the MT-based stalk-like structures in stage VI tubules but considerably diminished in stage VII tubules. At stage VI, formin 1 was robustly expressed at the apical ES and basal ES/BTB, colocalized with β-tubulin and EB1. At stage VII, although formin 1 remained prominently expressed at the apical ES and colocalized with β-tubulin and EB1, its expression considerably reduced at the basal ES/BTB. Cell nuclei were visualized by DAPI (blue). Scale bar, 40 μm, which applies to other micrographs of the same experiment. It is noted these findings are representative findings of an experiments which was repeated 3 times using different rat testes and yielded similar results. D, Lysates of rat testes were used for Co-IP, illustrating structural interaction between formin 1 and α-tubulin (left panel), formin 1 and EB1 (middle panel), as well as EB1 and α-tubulin (right panel) vs normal mouse IgG serving as negative controls in corresponding experiments. Interactions between formin 1 and β-actin vs EB1 and α-tubulin served as the corresponding positive controls. IgG, both the heavy and light chains, shown here served as the IgG input control. Data shown here are representative results of an experiment with n = 3 experiments which yielded similar results.