Additional file 1 of HMGB3 promotes the malignant phenotypes and stemness of epithelial ovarian cancer through the MAPK/ERK signaling pathway

Additional file 1

The <i>MTDH</i> (−470G>A) genotype & Allele distribution in Ovarian Cancer and controls.

†<p>The X² for HWE of Ovarian Cancer group and control group is 0.1 and 3.94 respectively (both <i>P</i>>0.05).</p

Results of association analysis between rs16896059 and clinicopathological characteristics.

<p>Results of association analysis between rs16896059 and clinicopathological characteristics.</p

Sequencing chromatograms of <i>MTDH</i> (−470G>A).

<p>A–C, the sequencing chromatogram results of the genotype GG, GA and AA respectively. Samples were chosen randomly.</p

Association of the −470G>A genotype and <i>MTDH</i> (−470G>A) protein expression.

<p>A, Relative level of MTDH protein expression in ovarian cancer tissues compared to normal ovarian tissues. B, Relative level of MTDH protein expression in the ovarian cancer tissues of patients with different −470G>A genotypes. C, Relative level of MTDH protein expression in normal tissues of individuals with different −470G>A genotypes. One circle represents the mean of three independent measurements from one patient. The distribution of the three genotypes were random between the groups. N represents the samples number of respective group. Bars represent the standard deviation. Student’s t test was used to evaluate the differences in the expression levels of different constructs.</p

Monocyte Chemoattractant Protein-1 Secreted by Decidual Stromal Cells Inhibits NK Cells Cytotoxicity by Up-Regulating Expression of SOCS3

<div><h3>Background</h3><p>Decidual stromal cells (DSCs) are of particular importance due to their pleiotropic functions during pregnancy. Although previous research has demonstrated that DSCs participated in the regulation of immune cells during pregnancy, the crosstalk between DSCs and NK cells has not been fully elucidated. To address this issue, we investigated the effect of DSCs on perforin expression in CD56⁺ NK cells and explored the underlying mechanism.</p> <h3>Methodology/Principal Findings</h3><p>Flow cytometry analysis showed perforin production in NK cells was attenuated by DSC media, and it was further suppressed by media from DSCs pretreated with lipopolysaccharide (LPS). However, the expression of granzyme A and apoptosis of NK cells were not influenced by DSC media. ELISA assays to detect cytokine production indicated that monocyte chemoattractant protein-1 (MCP-1) in the supernatant of DSCs conditioned culture significantly increased after LPS stimulation. The inhibitory effect of DSC media on perforin was abolished by the administration of anti-MCP-1 neutralizing antibody. Notably, reduced perforin expression attenuated the cytotoxic potential of CD56⁺NK cells to K562 cells. Moreover, Suppressor of cytokine signaling 3 (SOCS3) expression in NK cells was enhanced by treatment with MCP-1, as measured by RT-PCR and western blot. Interestingly, MCP-1-induced perforin expression was partly abolished by the siRNA induced SOCS3 knockdown. Western blot analysis suggested that both NF-κB and ERK/MAPKs pathway were involved in the LPS-induced upregulation of MCP-1 in DSCs.</p> <h3>Conclusions/Significance</h3><p>Our results demonstrate that LPS induces upregulation of MCP-1 in DSCs, which may play a critical role in inhibiting the cytotoxicity of NK cells partly by promoting SOCS3 expression. These findings suggest that the crosstalk between DSCs and NK cells may be crucial to maintain pregnancy homeostasis.</p> </div

The effects of DSCs media on apoptosis and expression of perforin and GrzA in NK cells.

<p>Peripheral CD56⁺ NK cells were incubated with fresh complete media, DSC-conditioned media or DSC-LPS media (prepared as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041869#s4" target="_blank">Materials and Methods</a></i>) for 48 hours. The apoptosis and intracellular expression of perforin and GrzA were examined by flow cytometry. (A) Flow cytometric analysis of perforin-positive cells. (B) GMean value of perforin expressed in CD56⁺ NK cells exposed to different types of media. (C) The percentage of GrzA-positive cells. (D) Flow cytometric analysis the apoptosis rate of CD56⁺ NK cells. The data shown represent three independent experiments (mean±SD), and the images are from an experiment that was representative of three independently conducted experiments. *<i>P</i><0.05 and *** <i>P</i><0.0001.</p

The frequency of Tc17 cells by flow cytometry.

<p>(a) Tc17 frequencies in the three groups. Significantly increased Tc17 cells were found in untreated UCC patients (<i>P</i> = 0.0076, n = 49) and CIN patients (<i>P</i> = 0.0149, n = 25) compared to healthy controls (n = 28). (b) Tc17 frequency in positive or negative lymph node metastases in UCC patients. Compared with patients without lymph node metastases (n = 15), significantly increased Tc17 frequency (<i>P</i> = 0.0026) was found in lymph node metastases patients (n = 35). Student's t test was used and bars represent SD, *<i>P</i><0.05, **<i>P</i><0.01.</p

Clinical Characteristics of CIN Patients.

<p>Clinical Characteristics of CIN Patients.</p

Analysis of Dll4 using immunohistochemistry.

<p>The protein expression of Dll4 was up-regulated in missed abortion (A) than that in induced abortion (B).</p