Expression of epithelial, fibroblast and endometrial markers in the primary epithelial and fibroblast cell cultures.

<p>Total RNA was subjected to quantitative real-time PCR analysis of epithelial markers (EpCAM, cytokeratin 8, E-cadherin), fibroblast markers (alpha-smooth muscle actin (αSMA), vimentin) (<b>A</b>–<b>C</b>), estrogen receptor 1 and 2 (ER1, 2), progesterone receptor (PR), progestagen-associated endometrial protein (PAEP) and matrix metalloproteinase 1 and 9 (MMP1, 9) (<b>D</b>–<b>F</b>). Data, average; error bars, S.E.M. Data shown are representative of three independent experiments.</p

Establishment of epithelial and fibroblast cells from human endometrial cancer tissues.

<p>Hematoxylin & eosin (H and E) staining of tissues and phase contrast images of established primary cells isolated from endometrial cancer: EC6 (<b>A</b>), EC14 (<b>B</b>), EC7 (<b>C</b>) and EC11 (<b>D</b>). Magnification: 100x. Subsequently these were digested with collagenase and cultured, prior to epithelial and fibroblast cell isolation using CD326 (EpCAM) and anti-fibroblast labeled magnetic beads.</p

Role of PI3K/Akt and MAPK/Erk signaling pathways in cancer-associated fibroblast-mediated endometrial cell proliferation.

<p>(<b>A</b>) Western blot analysis of phosphorylated-Akt (Ser473) and -Erk protein expression in ECC-1 cells after treated with either normal endometrial fibroblast T-HESC cells or cancer-associated fibroblast cells (EC6-Fib, EC7-Fib, EC11-Fib and EC14-Fib). Densitometry analysis compared the relative expression level of p-Akt and p-Erk to their total protein level. (<b>B</b>) Quantitative analysis of phosphorylated-Akt (Ser473 and Thr308) and phosphorylated-Erk levels in ECC-1 cells after treated with either T-HESC or CAFs, in comparison to cells treated with control (media containing 2% FBS). ECC-1 (<b>C</b>) and EC6-Ep (<b>D</b>) cells were treated with either PI3K pathway selective inhibitor (LY294002) or Erk pathway selective inhibitor (U0126) in the presence of cancer-associated fibroblasts conditioned media (1 µg/µl) for 72 hours. Data shown are cell viability after normalized with control (media containing 2% FBS). Data, average; error bars, S.E.M. *, <i>P</i><0.05; **, <i>P</i><0.0001. Data shown are representative of two independent experiments.</p

Fibroblast isolated from endometrial hyperplasia tissue did not promote endometrial cancer cell proliferation.

<p>Hematoxylin and eosin staining (<b>A</b>) and phase contrast image (<b>B</b>) of fibroblast cells isolated from human atypical hyperplasia tissue (EH-Fib); Magnification, 100x. (<b>C</b>) Flow cytometry analysis of EH-Fib stained with epithelial marker CD326-Alexa Fluor 647 and fibroblast marker CD90-PE. (<b>D</b>) Cell viability assay determining the effects of EH-Fib conditioned media on endometrial cancer cell lines (ECC-1 and HEC-1A) and primary epithelial cells (EC6-Ep and EC14-Ep) after 72 hours treatment. Data, average; error bars, S.E.M. *, <i>P</i><0.01. Data shown are representative of three independent experiments.</p

Differential effects of normal and cancer-associated endometrial fibroblast on EC cell proliferation.

<p>ECC-1 (<b>A</b>) and HEC-1A (<b>B</b>) cell lines and EC6-Ep (<b>C</b>) and EC14-Ep (<b>D</b>) primary endometrial cancer cells were tested with conditioned media prepared from cancer-associated fibroblasts (EC6-Fib, EC7-Fib, EC11-Fib and EC14-Fib) and normal endometrial fibroblast cell line (T-HESC) for 72 hours. Cell viability was examined using MTT assay and were normalized with control (media containing 2% FBS). CAFs shown are the average of all the four cancer-associated fibroblasts tested. Data, average; error bars, S.E.M. *, <i>P</i><0.005;. **, <i>P</i><0.0001. Data shown are representative of three independent experiments.</p

EpCAM and CD90 expression in established primary culture.

<p>Flow cytometry analysis of primary epithelial and fibroblast cells isolated from endometrial cancer tissues was performed after labeling cells with CD326 antibody-conjugated with AlexaFluor647 and CD90 antibody-conjugated with PE. Epithelial endometrial cell line, ECC-1 (<b>A</b>), primary epithelial cells EC6-Ep (<b>B</b>) and EC14-Ep (<b>C</b>), normal endometrial stromal cell line, T-HESC (<b>D</b>) and primary fibroblast cells, EC6-Fib (<b>E</b>), EC7-Fib (<b>F</b>), EC11-Fib (<b>G</b>) and EC14-Fib (<b>H</b>).</p

Mechanism of action of rapamycin as PI3K/mTOR pathway inhibitor.

<p>ECC-1 cell line (<b>A</b>) and EC6-Ep primary epithelial cell (<b>B</b>) were treated with increasing dose of rapamycin for 72 hours under the influence of control media (media containing 2% FBS) or 1 µg/µl EC11-Fib conditioned media. (<b>C</b>–<b>E</b>) ECC-1 cells treated with 2 µM of rapamycin with or without 1 µg/µl EC11-Fib conditioned media for 72 hours, were stained with annexin V-PE and 7-AAD before analyzed with flow cytometry. Data, average; error bars, S.E.M. *, <i>P</i><0.0001 compared to EC11-Fib treated cells. Data shown are representative of two independent experiments.</p

Characteristics of human immunodeficiency virus (HIV)-infected Malaysians included in this study.

<p>Characteristics of human immunodeficiency virus (HIV)-infected Malaysians included in this study.</p

Risk factors associated with CD4 T-cell recovery between the 0 to 12 month period following ART initiation.

<p>Risk factors associated with CD4 T-cell recovery between the 0 to 12 month period following ART initiation.</p

Correlation of K/T ratio with IL-6, IFN-γ, neopterin, hsCRP, sCD14, HSV-1 antibody level, HSV-2 antibody level, CMV antibody level and VZV antibody level.

<p>Correlation of K/T ratio with IL-6, IFN-γ, neopterin, hsCRP, sCD14, HSV-1 antibody level, HSV-2 antibody level, CMV antibody level and VZV antibody level.</p