Quantitation of expression of PTN and ESR1 in normal and cancerous ovaries from hens.

<p>[A] RT-PCR analyses were performed using cDNA templates from normal and cancerous ovaries of laying hens using chicken <i>PTN</i> and <i>ACTB</i>-specific primers. Lanes 1 to 4 show results of analyses of four normal ovaries. [B] Lanes 1–9 are from analyses of 9 different cancerous ovaries from laying hens. Expression of <i>PTN</i> mRNA was abundant in all carcinomas, but not in normal ovaries. Legend for panel B: Lane 1, endometrioid/serous/mucinous carcinoma (Stage III); Lane 2, endometrioid carcinoma (Stage I); Lane 3, serous carcinoma (Stage I); Lane 4, mucinous/endometrioid carcinoma (Stage IV); Lane 5, endometrioid carcinoma (Stage IV); Lane 6, endometrioid carcinoma (Stage III); Lane 7, clear cell carcinoma (Stage IV); Lane 8, serous/mucinous carcinoma (Stage IV); and Lane 9, serous/mucinous/endometrioid carcinoma (Stage III) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034215#pone.0034215-Lim1" target="_blank">[21]</a>. [C] The q-PCR analysis for <i>PTN</i> mRNA was performed using cDNA templates from normal and cancerous ovaries of laying hens (mean ± SEM; P<0.001). [D] <i>In situ</i> hybridization analyses of <i>PTN</i> mRNA in normal and cancerous ovaries of hens. Cross-sections of normal and cancerous ovaries of hens hybridized with antisense or sense chicken <i>PTN</i> cRNA probes demonstrated abundant <i>PTN</i> mRNA predominantly in GE of cancerous ovaries, but not in LE, stroma or blood vessels. [E] Immunoreactive PTN protein in normal and cancerous ovaries of hens. For the IgG control, normal rabbit IgG was substituted for the primary antibody. Sections were not counterstained. Legend: GE, glandular epithelium. <i>Scale bar</i> represents 200 µm (the first horizontal panels, sense and IgG) or 50 µm (the second horizontal panels, sense and IgG). [F] The q-PCR analysis for expression of <i>estrogen receptor alpha (ESR1)</i> was performed using cDNA templates from normal and cancerous ovaries of laying hens (mean ± SEM; P<0.001). [G] Immunoreactive ESR1 protein in normal and cancerous ovaries of hens. For the IgG control, normal rabbit IgG was substituted for the primary antibody. Sections were not counterstained. Legend: GE, glandular epithelium. <i>Scale bar</i> represents 200 µm (the first horizontal panels, sense and IgG) or 50 µm (the second horizontal panels, sense and IgG). <i>See </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034215#s4" target="_blank"><i>Materials and Methods</i></a><i> for a complete description of the methods</i>.