NF-κB activation enhances hTREX84 expression in immortal and/or cancer cells.

<p><i>A</i>, Schematic diagram of the hTREX84 promoter indicating the conserved NF-κB DNA binding motif. <i>B</i>, ChIP assay of RelA/p65 binding to <i>hTREX84</i> gene promoter in MDA-MB-231 (lane 1, 2); OVCAR5 (lane 3, 4); OVCAR 10 (lane 5, 6). Cells were cultured for 48 h. ChIP assays were then performed with anti-RelA/p65 antibody. PCR analysis was performed on immunoprecipitation samples without antibody (lane 1, 3, 5), with RelA/p65 antibody (lane 2, 4, 6). <i>C</i>, MCF-10F cells were transiently transfected with a control vector (lane 1) or a RelA/p65 cDNA expression construct for 48 hours. Western blot analysis for RelA/p65, hTREX84 and β-actin. <i>D</i>, Western blot analysis of RelA/p65, hTREX84 and β-actin protein levels after treatment of MDA-MB-231 cells with control siRNA (lane 1) and siRNA against RelA/p65 (lane 2) for 72 hours.</p

RelA/p65 expression in human normal breast tissue and tumors.

<p>RelA/p65 expression in human normal breast tissue and tumors.</p

hTREX84 is aberrantly expressed in ovarian cancer cells.

<p><i>A,</i> hTREX84 protein expression in representative ovarian cancer cell lines (OVCAR10, UPN251, UPN275, UPN289), immortal epithelial cell lines (HIO-118, HIO-102, HIO-104, HIO-113), primary epithelial cells (ROE). Protein samples were separated on a SDS-polyacrylamide gel immunoblotted using anti-hTREX84 or ß-actin monoclonal antibodies. <i>B,</i> hTREX84/ß-actin ratio in primary ovarian epithelial cell cultures (epithelial), immortal epithelial cell lines (HIO) and cancer cell lines (cancer).</p

Sodium bisulfite DNA sequencing of CpG sites in the <i>hTREX84</i> promoter and exon 1 regions.

<p><i>A</i>, DNA sequence of <i>hTREX84</i> regulator regions. CpG sites are shown in green color. Nucleotides are numbered on the right from the AUG translation start code which is underlined. <i>B</i>, Sodium bisulfite sequencing of DNA isolated from untreated (I) and treated (II) cells. The stars indicate CpG sites. <i>C</i>. Sodium bisulfite sequencing of DNA from a normal breast tissue (N) and an invasive ductal carcinoma (T). The stars indicate CpG sites.</p

HIO-107 cells were treated with 5-aza-dC to examine changes in DNA methylation in CpG sequences in <i>hTREX84</i>.

<p><i>A,</i> HIO-107 cells were treated with 5-aza-dC at concentrations of 1, 5, 10, 50 µM respectively for 5 days. RT-PCR show <i>hTREX84</i> mRNA expression and <i>B</i>, western blot analysis show hTREX84 protein levels. <i>C, D,</i> Quantitative mRNA and Western blot data were calculated from densitometric analysis of bands with the NIH imageJ software, respectively. The values were normalized to β-actin as internal control.</p

Determination of <i>hTREX84</i> promoter activities in MCF-10F cells with hTREX84/pGL3 reporters.

<p><i>A,</i> DNA sequence of NF-κB1M demonstrating that the first NF-kB binding site is mutated from 5′-GGAAACTCCC-3′ to 5′-CCAAACTCCC-3′. <i>B,</i> DNA sequence of NF-κB2M, demonstrating that the second NF-kB binding site is mutated from 5′-AGGTAATCCA-3′ to 5′-ACCTAATCCA-3′. N5-κB1/2M represented both of the two NF-κB binding sites in hTREX84 promoter region were mutated as described above (sequence not shown). <i>C</i>, Promoter activities among the three reporters constructs containing either a single mutated NF-κB binding sites or both (NF-κB1/2M) as determined by a luciferase assay. 1) Wild type NF-κB binding sites; 2) NF-κB1M; and 3) NF-κB2M; and 4) NF-κB1/2M.</p

Depletion of hTREX84 leads to defects in cellular proliferation of OVCAR10.

<p><i>A,</i> Analysis of hTREX84 and GAPDH mRNA levels following treatment of cells with siRNA against hTREX84 or control siRNA. <i>B,</i> Analysis of hTREX84 and β-actin protein levels after treatment of cells with siRNA against hTREX84 or control siRNA. <i>C,</i> Analysis of hTREX84 expression following siRNA treatment for 72 hours by immunofluoresence staining in the cells (<i>left,</i> cells transfected with control siRNA; <i>right,</i> cells treated with hTREX84-siRNA). <i>D,</i> Photomicrographs showing the morphology following depletion of hTREX84 (<i>left,</i> tumor cells transfected with control siRNA; <i>right,</i> cells treated with hTREX84-siRNA). <i>E,</i> Cell proliferation assay of tumor cells following depletion of <i>hTREX84</i>. Cell proliferation and apoptosis (data not shown) was examined using Guava ViaCount and Nexin assays respectively. The number of viable cells (x10⁴) are plotted against treatment duration at 24, 48, and 72 hrs after treatment with control siRNA or with hTREX84-siRNA. Shown are the results of three independent experiments. The difference is statistically significant. *, p<0.05; **, p<0.01. <i>F</i>. FACS analysis of the cells following down-regulation of hTREX84 levels. Shown is the percentage of cells in G1, S, G2-M after 72 hour of treatment with either siRNA (left panel) or hTREX84-siRNA (right panel).</p

Immunohistochemical staining of RelA/p65 proteins in representative breast cancer tissue specimens.

<p><i>A</i>, RelA/p65 was weakly detected in normal breast epithelial cells and positive products were located in the cell cytoplasm. <i>B</i>, Staining for RelA/p65 was detected mainly in the cytoplasm in high differentiated tumors. <i>C</i>, Intense and distinctly granular staining for RelA/p65 was detected in stained nuclei of low differentiated tumor specimens. <i>D</i>, Tumor section evaluated without the primary antibody to serve as a negative control. Magnification 200x.</p

Analysis of expression of MET-associated proteins.

<p>Protein expression of the epithelial-associated markers, e.g., mucin-1, CK-19 and occludin were evaluated by immunoblotting and band densities were quantified by Image-J software. Fold increases in protein expression of treated (20 µg/ml epimorphin) versus untreated A1847: 2.5-fold increase for CK-19; 1.5-fold increase for mucin-1, and 4.5-fold increase for occludin. β-actin served as the loading control. (means ± S.D., n = 3). [*<i>p</i><0.05 compared with control].</p

β-catenin activation of A1847 in response to epimorphin.

<p>Untreated and 20 µg/mL epimorphin-treated A1847 and OVCAR10 were analyzed for β-catenin, a marker of epithelial differentiation, by immunostaining. β-catenin (green), DAPI (blue) and merge (neon green) in untreated A1847 (A–C) and OVCAR10 (G–I); epimorphin-treated A1847 (D–F) and OVCAR10 (J–L). Immunostaining analysis showed increased expression of β-catenin-positive cells in epimorphin-treated A1847 (D&F) and epimorphin-treated OVCAR10 (J&L). There were abundant β-catenin positive cells at and around the cell-cell junctions of epimorphin-induced A1847 and OVCAR10 (F&L) compared to untreated controls (C&I).</p