Detection of hPL protein in overexpressing, but not in control breast cancer cells by mAb-4 and mAb-6 monoclonal antibodies.

<p>(A) Whole cell lysates from breast cancer cells, non-tumorigenic breast epithelial cells and choriocarcinoma cells were subjected to western blotting; hPL was detected using mAb-4 and mAb-6; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087325#pone-0087325-g002" target="_blank">Figure 2</a> for other details. (B) Real-time PCR was used to compare overexpression of CSH mRNA in lentiviral-infected breast cancer cell lines. Values (means±SEM; N = 3) represent fold increases over corresponding non-infected cells. (C) Whole cell lysates from control and PL-overexpressing cells were subjected to western blotting. hPL was detected using mAb-6 under the same conditions as in (A).</p

Immunohistochemical staining of human breast tumors using mAb-1 and mAb-12 monoclonal antibodies against hPL.

<p>Term placenta (top) showing specific staining of the syncytial layer by both antibodies. Representative examples of staining-negative normal tissue and staining-positive and -negative tumors are shown. All normal tissues tested were negative and some, but not all tumors, were positive.</p

Five different antibodies detect an hPL-like protein (‘hPL’) in breast cancer and choriocarcinoma cell lines by western blotting.

<p>Whole cell lysates from four breast cancer, two non-tumorigenic breast epithelial, and two choriocarcinoma cells were subjected to western blotting. hPL was detected using commercially available (mAb-1 and Sheep), and custom-made (Poly-1, Poly-2 and Poly-3) antibodies. 40 ug of protein was loaded for each cell line; 0.5 ug of placental lysate and 2 ng rhPL were loaded as positive controls. β-actin served as a loading control.</p

Expression of CSH mRNA in some primary breast carcinomas but undetectable hPL protein.

<p>(A) Comparison of CSH expression by real-time PCR in eight primary tumors (T1–T8) and three normal breast tissue samples (N1–N3). Data are presented as fold over ZR75 cells used for reference. (B) None of the above tissues show hPL protein, as determined in western blotting using mAb-6, which detected rhPL, placental hPL, and hPL in overexpressing MCF7 cells (MCF7-PL).</p

The CSH gene is expressed at variable levels in breast cancer and choriocarcinoma cell lines.

<p>(A) Expression of CSH mRNA in 5 breast cancer cells lines, 2 non-tumorigenic breast epithelial cells (HME and MCF10) and 2 choriocarcinoma cell lines (JAR and BeWo) as determined by real-time PCR. Data are expressed as fold change over HME after correction for β-actin (means±SEM; N = 3). (B) CSH products of identical sizes are produced in placenta, breast cancer and choriocarcinoma cell lines. The products were generated by using 30 cycles of conventional PCR, followed by electrophoresis on a 1% agarose gel and visualization with ethidium bromide.</p

Antibodies for hGH and hPRL do not detect ‘hPL’.

<p>Western blots with 40 µg of MCF7 breast cancer cell lysate, 5 ng of recombinant hPL, hPRL and hGH and 0.5 µg of placental lysate were probed with antibodies for hPL, hPRL and hGH.</p

Multiple antibodies against hPL are non-specific.

<p>(A) Conventional PCR showing efficient knockdown (KD) of CSH in MCF7 and ZR75 following transfection with shRNA. The placenta was used as a positive control and size marker. (B) Western blots with five different antibodies showing an unchanged intensity of the bands previously believed to be ‘hPL’ in both control and knock-downed (KD) cells. (C) Similar intensity of immunocytostaining in control and knockdown ZR75 cells using mAb-1 and mAb-12.</p