5 research outputs found
Additional file 1: Figure S1. of Aspirin inhibits epithelial-to-mesenchymal transition and migration of oncogenic K-ras-expressing non-small cell lung carcinoma cells by down-regulating E-cadherin repressor Slug
p53 mutation exerts increased migratory effect in combination with oncogenic K-ras-expressing system on NSCLC cells migration. Phase contrast images (left panels) depicting migration of NCI-H522 cells (oncogenic K-ras/mutant p53), mutant p53-reconstituted H1299 cellsĀ (wild type K-ras/mutated p53), A549 cells (oncogenic K-ras/wild type p53), and wild type p53 reconstituted H1299 cells (wild type K-ras/wild type p53). Graphical representation of percent cell migration in wound healing assay (right panel) with inset showing immunoblot analysis for the transfection efficiency of p53 - R175H clone in H1299 cells and p53 - cDNA in H1299 cells. Scale bar: 100Ā Ī¼m. (TIFF 2457Ā kb
Stat-5A transfection confers resistance to CD4<sup>+</sup> T cells from tumor-induced death.
<p>A, Stat-5A (<i>left panel</i>) and Stat-5B (<i>right panel</i>) isoforms were immunoprecipitated from cell lysates using specific antibodies and then Western blotted with anti-phospho-tyrosine or anti-Stat-5A/Stat-5B antibodies to determine phosphorylation status of specific proteins. B, Jurkat T cells were transfected with control vector, wild-type <i>Stat-5A/Stat-5B</i>, C-terminal truncated <i>Stat-5A</i><sub>713</sub>/<i>Stat-5B</i><sub>718</sub> or constitutively active <i>Stat-5A1*6</i> genes and were cultured in the presence of media alone or MCF-7 spent media (Ā±theaflavins) for 48 h. Percent cell death (Annexin-V-PE<sup>+</sup>/7AAD<sup>+</sup>) was determined flow cytometrically. Values are meanĀ±S.E.M. of three independent sets of experiments.</p
Cell free tumor supernatant leads to CD4<sup>+</sup> T cell depletion by inducing apoptosis.
<p>A, Purified human peripheral CD4<sup>+</sup> T cells were cultured in the presence of media alone or cell-free MCF-7-spent media (Ā±theaflavins, doses from 6.25 Āµg/ml to 50 Āµg/ml). After 48 hours, viable cell numbers were scored by Trypan Blue exclusion method. B, Graphical representation of percent apoptosis of CD4<sup>+</sup> T cells (<i>left panel</i>) and Jurkat T cells (<i>right panel</i>). CD4<sup>+</sup> T cells labelled with Annexin V-PE and 7AAD were analyzed flow cytometrically. Annexin V/7AAD-positive cells were regarded as apoptotic cells. Values are meanĀ±S.E.M. of five independent sets of experiments.</p
Re-confirmation of PGE<sub>2</sub> as the molecule behind tumor-induced perturbation in CD4<sup>+</sup> T cell survival signaling.
<p>A, MCF-7 cells were treated with theaflavins or celecoxib or transfected with Cox-2-siRNA and the levels of Cox-2 and GAPDH (internal control) mRNA were determined by RT-PCR (<i>upper panel</i>). Western blot analysis was performed for the determination of levels of Cox-2 or Ī±-Actin (internal control) proteins (<i>middle panel</i>). In parallel experiments the amount of tumor-secreted PGE<sub>2</sub> in the cell-free supernatant was determined by ELISA (<i>lower panel</i>). B, Purified CD4<sup>+</sup> T cells were cultured in the presence of media alone or MCF-7-spent media (tumors were either pre-treated with 25 Āµg/ml theaflavins/3.5 ng/ml PGE<sub>2</sub>/50 ĀµM celecoxib or transfected with 300pmole Cox-2-siRNA) for 48 h. Expression levels of IL2RĪ³c and Bcl-2 as well as phosphorylation status of Jak-3/-Stat-5 were determined by Western blotting in which Ī±-Actin was used as internal control (<i>upper panel</i>). In parallel experiments, flowcytometric determination of percent cell death (<i>lower panel</i>) was established. Values are meanĀ±S.E.M. of three independent experiments.</p
Tumor-shed PGE<sub>2</sub> is responsible for CD4<sup>+</sup> T cell apoptosis.
<p>A, Tumor-secreted PGE<sub>2</sub> over time in cell-free spent media of MCF-7 cells (control (ā), Cox-2-siRNA-transfected (Ī) or theaflavin-treated (ā¢) was determined by ELISA. B, Percent CD4<sup>+</sup> T cell death (Annexin-V-PE<sup>+</sup>/7AAD<sup>+</sup>), induced by the spent media as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007382#pone-0007382-g002" target="_blank">Fig. 2A</a>, was plotted over time. Values are meanĀ±S.E.M. of three independent sets of experiments.</p