Figure 1: Overexpression of p-rpS6-MT versus rpS6-WT and control (empty) vector in Sertoli cell epithelium with an established TJ-barrier on the Sertoli cell tight junction (TJ)-permeability barrier function. (a) Regimen used in this and subsequent experiments reported herein. Data presented herein were results of a representative experiment from three independent experiments, excluding pilot experiments used to establish experimental conditions, using different batches of Sertoli cells, which yielded similar results. (b) Composite data of three experiments based on findings shown in c, illustrating considerable increase in expression of rpS6 following overexpression of p-rpS6-MT and rpS6-WT versus empty vector in control Sertoli cells when rpS6 (green fluorescence) was quantified. Each bar is a mean ± standard deviation of three experiments. For each experiment, at least 50 Sertoli cells were randomly selected for fluorescence intensity analysis by Image J. *P < 0.01, Student's t-test by comparing cells from treatment group to control group with the fluorescence intensity of control cells arbitrarily set at 1 for statistical comparison. (c) Staining of cells with rpS6 (green fluorescence), red fluorescence represents Cy3-labeled plasmid DNA to confirm successful transfection. Sertoli cell nuclei were visualized by DAPI (blue). Scale bar = 40 μm, which applies to other micrographs in this panel. (d) Overexpression of rpS6 (pCI-neo/rpS6-WT) and the constitutively active quadruple phosphomimetic mutant (pCI-neo/p-rpS6-MT) versus controls (emptor vector, pCI-neo/Ctrl) were also confirmed by immunoblot analysis when the protein steady-state levels of rpS6 and its phosphorylated/activated forms were quantified (see bar graphs in the lower panel). A considerable down-regulation of mTOR was noted. Expression of many BTB-associated proteins that were examined was found not to be affected following overexpression of either rpS6-WT or rpS6-MT versus control groups. However, overexpression of rpS6-WT or rpS6-MT led to a down-regulation on the expression of p-Akt1-T307, p-Akt1-S473 and p-Akt2-S474 but not the total Akts (i.e., Akt1/2/3) (bar graphs in lower panel), also consistent with earlier reports., Bar graphs on the right panel represent composite data of three experiments. *P < 0.01, Student's t-test by comparing the treatment with control group wherein protein expression in control group was arbitrarily set at 1 for statistical comparison. [Supplementary Figure 1] for uncropped blots. (e) A study to assess changes in the Sertoli cell TJ-permeability barrier function by quantifying transepithelial electrical resistance (TER) across the Sertoli cell epithelium. This is the result of a representation experiment; each data point is the mean ± standard deviation of quadruple bicameral units from three independent experiments using different batches of Sertoli cells. *P < 0.05 and **P < 0.01, comparing each data point with the corresponding control by Student's t-test. DAPI: 4',6-diamidino-2-phenylindole; BTB: blood-testis barrier; mTOR: mammalian target of rapamycin; WT: wild type; Ctrl: control; MT: mutant.