Determination of EMT in HBCEC and HMEC.

<p><b>a</b>, Microscopic analysis of HMEC and various HBCEC after stimulation with TGF-β1 (10 ng/ml) for 48 h. P3 = passage 3. Bar, 100 µM. <b>b</b>, Expression of the indicated EMT-associated marker genes in the same HMEC and HBCEC shown in <b>a</b>. Data represent the mean ± SD of three reactions after normalization with the housekeeping genes TBP and β-actin.</p

RTCA migration assay with Tu459 NSCLC cells, HBCEC, and HMEC.

<p>A total of 50,000 cells of each population were seeded per well in the upper chamber of a CIM-Plate 16. Cells were left untreated or were stimulated with TGF-β1 (10 ng/ml). The three HBCEC were compared to the same HMEC. Data shown represent the mean ± SD of three-four wells processed in parallel. For Tu459 cells, HBCEC #335 and #699 a representative experiment from 3 experiments is shown, for HBCEC #338 an experiment with data intermediate between those of two other experiments run with identical conditions is presented. Differences were highly significant (p<0.001) at the 8, 16, 24, 32, and 40 h time points between untreated HBCEC (red curves) and untreated HMEC (blue curves).</p

Expression of MMP-2, uPA and PAI-1 in HBCEC, HMEC, and permanent breast cancer cell lines.

<p><b>a</b>, QPCR determination of MMP-2 expression in MDA-MB-231, MDA-MB-435s, and MCF-7 cells (upper graph) and in HMEC and various HBCEC (lower graph) after stimulation with TGF-β1 (10 ng/ml) for 24 h and 48 h. Data are presented as mean ± SD of three reactions after normalization with the housekeeping genes TBP and β-actin. The numbers above the graphs indicate -fold induction by TGF-β1 relative to unstimulated controls. Asterisks indicate significant differences relative to the respective treatment groups in HMEC. <b>b</b>, Expression of uPA (upper graphs) and PAI-1 (lower graphs) in MDA-MB-231 and MCF-7 cells (left graphs), and HMEC and HBCEC (right graphs) as measured by ELISA. Data represent the mean ± SD of duplicate determinations. (n = 2 to 3). n.d., not detectable.</p

Real-time measurement of cell migration and cell invasion in various established breast carcinoma cell lines using the xCELLigence DP system.

<p><b>a</b>, left panel, Migration analysis of MDA-MB-231, MDA-MB-468, and MCF-7 cells. Cells were stimulated with either TGF-β1 (5 ng/ml) or EGF (20 ng/ml) as indicated by the color code in serum-reduced medium during the assay. At the 8, 16, 24, 32, and 40 h time points differences were highly significant (p<0.001) between control (red curve) and TGF-β1-treated (blue curve) but not between control and EGF-treated (cyan curve) MDA-MB-231 cells. Right panel, Migration curves of MDA-MB-435s cells in the presence of the ALK5 inhibitor SB431542 (5 µM) or vehicle-only (0.05% DMSO). At the 16, 24, 32, and 40 h time points differences were highly significant (p<0.001) between SB431542+TGF-β1-treated (pink curve) and vehicle+TGF-β1-treated (green curve) MDA-MB-435s cells. <b>b</b>, Matrigel invasion RTCA assay with MDA-MB-231 cells (left panel) and MDA-MB-435s cells (right panel) with or without TGF-β1 stimulation (5 ng/ml). Both cell lines were treated with TGF-β1 in the presence of either vehicle-only (0.05% DMSO) or the ALK5 inhibitor SB431542 (5 µM). In order to prepare the CIM-Plates 16 for the invasion setup, the bottom of the upper compartment of each well was covered with a thin layer of Matrigel prior to cell seeding. Data are depicted as mean ± SD of three or four wells processed in parallel. In each panel, a representative experiment is shown. At the 8, 12, 16, and 20 h time points differences were highly significant (p<0.001) between control (red curve) and TGF-β1-treated (green curve) MDA-MB-231 cells. Differences were significant (p<0.05) between SB431542+TGF-β1-treated cells (pink curve) and vehicle+TGF-β1-treated cells (green curve) at the 12, 16, and 20 h time points (MDA-MB-231, left panel) and at the 24, 32, and 40 h time points (MDA-MB-435s, right panel).</p

Additional file 1: of Dasatinib blocks transcriptional and promigratory responses to transforming growth factor-beta in pancreatic adenocarcinoma cells through inhibition of Smad signalling: implications for in vivo mode of action

Primers used for qRT-PCR. (DOC 51 kb

Cartoon to illustrate the effects of activins and TGF-βs, their receptors and target genes, and their inhibitors, on pluripotency and growth of adherently growing PCMO.

<p>Stimulatory (↓) and inhibitory (⊥) interactions are depicted by bold and dashed lines to indicate strong and weak interactions, respectively. Ab, antibody.</p

Activin A and TGF-β1 synthesis and secretion during conversion of monocytes to PCMO.

<p>(A) Standard endpoint RT-PCR-based detection for the β_A-subunit of activin and TGF-β1 in PCMO. MW marker, molecular weight marker = 100 bp ladder. (B) Activin A and TGF-β1 levels in culture supernatants of monocytes/PCMO growing adherently (a) or in supsension (s) at different time points during culture. Supernatants, conditioned for 24 h, were taken every day until day 4 and subjected to ELISA specific for activin A or total TGF-β1, and bioassay for TGF-β. Data were calculated after subtraction of background activin A and TGF-β levels (contained in the AB serum) and represent means ± SD from five different donors normalized to 10,000 cells. Asterisks indicate a significant difference relative to the respective levels on day 1. Numbers below the graph indicate the percentage of active TGF-β in the respective samples as determined by bioassay (means ± SD). Differences were significant between day 2 and day 4 in both adherent and suspended cells.</p

PCMO respond to exogenous activins and TGF-β1 with phosphorylation of Smad2.

<p>(A) PCMO were stimulated on day 4 of culture with 50 ng/ml of either activin A, activin B, or activin AB in the presence or absence of the ALK4/5/7 inhibitor SB431542 (1 μM), the p38 MAPK inhibitor SB203580 (10 μM), vehicle (dimethylsulfoxide, 0.1%), or medium alone (w/o) for 1 h followed by immunoblotting for phosphorylated Smad2C (p-Smad2), total Smad2 (Smad2), and β-actin as a loading control. (B) Responsiveness of PCMO from two different donors to TGF-β1 stimulation. PCMO were stimulated on day 4 with 5 ng/ml TGF-β1 in the presence or absence of SB431542 (1 μM), or vehicle for 1 h followed by immunoblotting for p-Smad2C and Smad2. Data in A and B are representative of four different donors.</p

Identification of the autocrine ligands that regulate pluripotency gene expression and proliferation in PCMO.

<p>Effect of SB431542 (SB), follistatin (FS), and anti-TGF-β antibody (α-TGF-β) on day 4 of culture on (A) Smad2C phosphorylation as determined by immunoblotting and ELISA (graph below blot). Co, isotype control. Numbers in brackets indicate the concentrations used (μM for SB and μg/ml for FS and α-TGF-β. (B) Oct4A and Nanog expression as determined by qPCR (graphs) and immunoblotting. Oct4 and Nanog were immunodetected in nuclear proteins (25 μg/lane) from the same day 3 (Oct4) and day 4 (Nanog) PCMO used for qPCR analysis. Successful enrichment of nuclear proteins and equal protein loading was verified with an antibody to histone H4. (C) Effect of FS, and α-TGF-β on Smad3C phosphorylation and p21^WAF1 expression in day 4 PCMO as determined by immunoblotting and ELISA (graphs below blots). (D) Ki67 expression on day 4 of culture. In (B) and (D), SB, FS, and α-TGF-β were used at concentrations of 1 μM, 0.1 μg/ml, and 5 μg/ml, respectively. ELISA data in A and C, and qPCR data in B and D are means ± SD from triplicate samples and were derived from one donor. Data are representative of four different donors. Asterisks indicate a significant difference between vehicle and SB or FS-treated cells and between Co and α-TGF-β-treated cells.</p

Expression of cell cycle regulating genes in PCMO following neutralization of endogenous activin(s) and TGF-β(s).

<p>QPCR-based detection of the indicated genes after treatment of PCMO cultures with follistatin or anti-TGF-β antibody. Data represent the-fold stimulation relative to untreated cells (follistatin) or isotype IgG1-treated control cells (anti-TGF-β antibody) on day 4 of culture, mean ± SD. Data (means ± SD from triplicate samples) are from one donor and are representative of four different donors. ANAPC2, anaphase promoting complex subunit 2; CDC2, cell division control protein 2; CDK, cyclin-dependent kinase.</p