Demonstration of elevated amounts of PIM1 and HMGA1 proteins in PECs after adherence by <i>T. vaginalis</i> (<i>Tv</i>).

<p>In this experiment, trichomonads were added to T25 confluent monolayers of PECs (lane labeled+) using a parasite to PEC ratio of 10∶1. PECs without added organisms are labeled with a minus sign (−). This ratio of 10∶1 was chosen because it has been shown to yield at least one parasite attached per epithelial cell in adherence assays and to optimally signal epithelial cells for up-regulation of expression of genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Kucknoor1" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Garcia1" target="_blank">[27]</a>. After visible attachment to PECs, non-adherent organisms were decanted and fresh PEC medium added followed by incubation at 37°C for an additional 30 min. The flask was then placed directly in ice for detachment of organisms, after which PECs were washed and removed from the flask for preparation of total proteins for immunoblotting using established protocols, polyclonal rabbit antibodies produced in our laboratories, and equal loading of protein onto gels, as detailed previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Under conditions of exposure of PECs with or without <i>T. vaginalis</i>, no change in the amount of other cellular proteins was detected, as evidenced by no changes in the amounts of Akt and Bad, and this served to show equal amounts of total proteins loaded onto gels for SDS-PAGE and immunoblotting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Prebleed rabbit serum was used as the negative control and gave no reactivity.</p

Summary of mice exhibiting neuroendocrine differentiation as established by positive synaptophysin staining.

<p>Summary of mice exhibiting neuroendocrine differentiation as established by positive synaptophysin staining.</p

HF/HS diet exposure stimulates increased proliferation in male offspring.

<p>(A) Representative pHH3 and Ki67 staining of prostates from Pten knockout mice exposed to maternal Chow or HF/HS diet Red—pHH3, Green—Ki67, Blue—DAPI (nuclei). Scale bars = 25 μm. (B and C) Quantification of pHH3- (B) and Ki67- (C) positive cells normalized to total number of cells per image. Same letters mean there’s no significant difference between conditions, different letters represent statistically significant differences. n = 10 images per mouse from n = 5 mice per experimental group; <i>P</i><0.05 as calculated by 2-way analysis of variance followed by Tukey’s multiple comparison’s test.</p

HF/HS diet exposure does not affect phospho-Akt and Akt expression in Pten knockout males.

<p>Representative images of phospho-Akt (Ser-473) (a-d), Akt (e-h) and Pten (i-l) staining of prostates from WT and Pten knockout mice exposed to maternal Chow or HF/HS diet. Red—Pten, phospho-Akt, Green—Akt, Blue—DAPI, scale bar = 50 μm.</p

Features of neuroendocrine differentiation observed in Pten kockout males.

<p>Representative H&E and synaptophysin staining of prostates from WT and Pten knockout. The WT and Pten knockout mice exposed to maternal HF/HS diet showed similar findings as their Chow counterparts and these findings are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175764#pone.0175764.t001" target="_blank">Table 1</a>. Black arrows show areas of NED. Scale bar = 100 μm.</p

Experimental set-up.

<p>Four-week-old Pten^loxp/loxp female mice were fed either a Chow or HF/HS diet for four weeks and then mated to Pten^loxp/loxp;PBCre4⁺ males. Male offspring were fed a Chow diet after weaning (three weeks) and until sacrifice at six weeks of age.</p

Summary of prostate cancer phenotypes in Pten knockout mice and WT littermate controls.

<p>Summary of prostate cancer phenotypes in Pten knockout mice and WT littermate controls.</p

Metabolic consequences of HF/HS diet in nine-week-old Pten^loxp/loxp dams.

<p>Dot plots show quantitation of body weight (A), percent fat mass (B), percent lean mass (C), percent water (D), glucose tolerance (E), and insulin sensitivity (F). n > 3 mice per group, *<i>P</i><0.05 as calculated by student’s t-test.</p