TMAs used for immunohistochemical analysis of LEDGF/p75 protein expression in tumors.

<p>TMAs used for immunohistochemical analysis of LEDGF/p75 protein expression in tumors.</p

LEDGF/p75 transcript expression in human tumors determined by analysis of Oncomine cancer gene microarray database.

<p>*P<0.05.</p

Elevated immunohistochemical expression of LEDGF/p75 protein in five tumor types compared to corresponding normal tissues.

<p>Tissue microarrays were stained with antibody against LEDGF/p75, and the individual cores were blindly scored using the following scale: 0 = no staining, 1 = low staining, 2 = moderate staining, 3 = strong staining. Scored tissues were pooled into two groups: low staining (scores 0 and 1, dark bars) and high staining (scores 2 and 3, light bars). The percentage of specimens in the two staining categories was plotted for tumor tissues compared to normal (including disease-free normal and normal adjacent) tissues. *<i>P</i><0.05; **<i>P</i><0.01. <i>P</i> values were determined with Fisher's exact test.</p

Association of LEDGF/p75 protein expression in colon, liver, prostate, thyroid and uterine tumors with patients' clinical characteristics.

<p>Bold numbers denote significant <i>P</i> values. <i>P</i> values for the association of immunohistochemical LEDGF/p75 expression (high vs. low comparison) in tumors with patients' age and sex were calculated using Fisher's exact test, while those for tumor stage were calculated using Kendall's tau b correlation analysis. At the time of surgical excision of the tissues, the median age of donors was 62 years for colon cancer, 53 years for liver cancer, 66 years for prostate cancer, 48 years for thyroid cancer, and 70 years for uterine cancer. The tumor stages (pT1 to pT4) correspond to pathologic tumor stages T1 through T4 used in the TNM system of cancer staging. The TNM system is based on the extent of the tumor (T), the extent of spread to the lymph nodes (N), and the presence of distant metastasis (M). A number is added to each letter (eg. T1, T2, T3, T4) to indicate the size or extent of the primary tumor and the extent of cancer spread. N/A, Not applicable; N/D, No data available.</p

Transcript expression of LEDGF/p75 in eight human cancer types determined by TissueScan Cancer Q-PCR analysis.

<p>Data were analyzed using the ΔΔCt method with values normalized to β-actin levels. The y-axis represents the induction fold of the LEDGF/p75 mRNA level in eight cancer types (n = 9) compared to matching normal adjacent tissues (n = 3) in the array. Error bars displays the range of standard error. * <i>P</i><0.05. <i>P</i> values were determined with Student's t-test.</p

Immunohistochemical staining for LEDGF/p75 protein in selected human tumors.

<p>A. Representative images of immunohistochemical staining (low intensity, scores 0–1; high intensity, scores 2–3) for LEDGF/p75 in prostate, colon, and thyroid tumors (Scale bar-40 µm; magnification = 200×). B. Representative images of LEDGF/p75 immunostaining of non-disease normal prostate and colon tissues, and in tumor tissues and their matched adjacent tissues (Scale bar-40 µm; magnification = 400×). TMAs were stained using LEDGF/p75 specific rabbit antibody Scripps-Ab5087, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030132#s2" target="_blank">Materials and Methods</a>. Identical camera settings were used in the acquisition and processing of images for a particular tissue type.</p

Differential expression of LEDGF/p75 protein analyzed by immunohistochemistry in 21 human tumor types and corresponding normal tissues.

<p>TMAs stained with antibody against LEDGF/p75 were scored as: 0 = no staining, 1 = low, staining, 2 = moderate staining, 3 = strong staining. Scored tissues were pooled in two groups: low staining (scores 0 and 1) and high staining (scores 2 and 3). <i>P</i> values comparing the immunohistochemical expression (high vs. low) of LEDGF/p75 protein between tumor tissues and corresponding normal tissues was determined using the Fisher's exact test. Bold numbers denote significant <i>P</i> values. n/a-Not applicable.</p

Identification of LEDGF/p75 specific antibody by immunoblotting in PC-3 cells with transient LEDGF/p75 knockdown.

<p>Cells were transfected with siLEDGF/p75 to induce transient knockdown of LEDGF/p75. PC-3 cells transfected with small interfering scrambled RNA duplex (siSD) served as corresponding control. Immunoblotting analysis tested the specific reactivity of all the antibodies against LEDGF/PSIP1. All the blot pairs (siSD and siLEDGF/p75) for each antibody were derived from the same blot.</p

Effects of transient depletion of LEDGF/p75 on ERp57 expression levels in DU145 cells.

<p>A. LEDGF/p75 and ERp57 transcript and protein levels were assessed by qPCR and immunoblotting, respectively. A. Transcript and protein expression levels of LEDGF/p75 and ERp57 in DU145-DR cells, selected for their resistance to DTX, compared to parental DU145 cells. B. Parental DU145 cells with and without siRNA induced transient depletion of LEDGF/p75. C. DU145-DR cells with and without siRNA induced transient depletion of LEDGF/p75. Each graph represents the average of at least 3 independent experiments performed in triplicates (*<i>P<0</i>.<i>05</i>, **<i>P</i> <0.01). <i>P</i> values were determined in comparison to cells transfected with non-specific, scrambled control siRNAs (siSD) using the Student’s <i>t</i>-test.</p

Multi-screen immunoblotting analysis to identify candidate stress proteins upregulated by LEDGF/p75 in RWPE-2 cells.

<p>A. Lysates from cells stably overexpressing LEDGF/p75 (RWPE-2–<i>ledgf/p75</i>) and RWPE-2 cells transfected with empty pcDNA3.1 vector (RWPE-2-<i>Vec</i>) were individually analyzed by immunoblotting using the Kinetworks™ KHSP-1.0 screen platform. Validation was performed using commercial antibodies and changes in protein expression were determined in LEDGF/p75-overexpressing RWPE-2 cells in comparison to cells transfected with empty vector. B. Untransfected RWPE-2 cells, cells transfected with empty vector, and cells stably overexpressing LEDGF/p75 were tested in-house for additional validation of upregulation of ERp57 and Hsp90. β-actin was used as loading control.</p