Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling-6

<p><b>Copyright information:</b></p><p>Taken from "Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling"</p><p>http://www.jmolecularsignaling.com/content/2/1/6</p><p>Journal of Molecular Signaling 2007;2():6-6.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1976611.</p><p></p>after induction of dnTCFs in LS174T derived cell lines. Expression levels were normalized to the Ubiquitin C (UBC) transcript. The CLU34 mRNA variant is specifically up-regulated in response to dnTCF1 over-expression in LS174T cells. Data are presented as the mean ± standard deviation from 2 separate experiments with each experiment consisting of the mean value of 3 independent determinations. c-MYC mRNA levels decrease, whereas p21mRNA levels increase after induction of both dnTCFs

Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling-1

<p><b>Copyright information:</b></p><p>Taken from "Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling"</p><p>http://www.jmolecularsignaling.com/content/2/1/6</p><p>Journal of Molecular Signaling 2007;2():6-6.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1976611.</p><p></p>the effectors of the Wnt pathway, were over-expressed in stably transfected LS174T derived cell lines. Proliferation was halted in LS174T derived cell lines after induction of both dnTCFs. This was visualised by methyl violet staining of cell cultures after 5 days of induction. Similar results were obtained by manually counting cells in a haemocytometer after cells had been cultured in the presence (+) or absence (-) of induction for 4 days. Data are presented as the mean ± standard deviation from 3 separate experiments. Induction of exogenous gene products, dnTCFs, was detected by western blot with cell lysates from LS174T derived cell lines 0, 12, and 24 hr after induction. Control cells are LS174T cells without any dnTCF expression vector. Both dnTCF1 and dnTCF4 abrogate β-catenin/TCF driven transcription of the Wnt-target gene, c-MYC, as analyzed by western blot with cell lysates from LS174T derived cell lines 0 and 12 hr after induction

Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling-0

<p><b>Copyright information:</b></p><p>Taken from "Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling"</p><p>http://www.jmolecularsignaling.com/content/2/1/6</p><p>Journal of Molecular Signaling 2007;2():6-6.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1976611.</p><p></p>f GFP tagged E-cadherin and GFP as control. Immunofluorescence was used to show the effects of transient over-expression of GFP tagged E-cadherin, which binds β-catenin, in LS174T and HCT116 colon carcinoma cell lines. In the left panel it is shown that the GFP-cyt-E-cadherin fusion protein efficiently sequesters β-catenin from the nucleus whereupon Wnt signaling is abrogated. In transfected cells (white arrow heads) β-catenin has a perinuclear localization contrasting untransfected cells (white arrows) where it is uniformly distributed. Shown in the right panel is the up-regulation of cytoplasmic CLU protein which follows the abrogation of Wnt signaling induced by the GFP-cyt-E-cadherin fusion protein. CLU is up-regulated in GFP-cyt-E-cadherin transfected cells (yellow arrow heads) but not in untransfected cells nor in cells transfected with GFP alone. Western blot with cell lysates from LS174T cells 24 hr after transient transfection with GFP-cyt-E-cadherin and GFP as a control. β-actin was used as a loading control. CLU protein levels are up-regulated and c-MYC protein levels are down-regulated as a consequence of over-expressing GFP-cyt-E-cadherin but not GFP alone. WB: western blot, ab: Antibody

Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling-5

<p><b>Copyright information:</b></p><p>Taken from "Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling"</p><p>http://www.jmolecularsignaling.com/content/2/1/6</p><p>Journal of Molecular Signaling 2007;2():6-6.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1976611.</p><p></p>ial TCF-binding sites (red arrows) predicted by the MatInspector software. Exonic structure of CLU mRNA variants

Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling-3

<p><b>Copyright information:</b></p><p>Taken from "Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling"</p><p>http://www.jmolecularsignaling.com/content/2/1/6</p><p>Journal of Molecular Signaling 2007;2():6-6.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1976611.</p><p></p>4T cells 48 hr after induction (p < 0.005, student's t-test). This was determined using two cell death assays based on either trypan blue dye or DNA-intercalating dyes, propidium iodide (PI) and hoechst 342 (HST). LS174T control and dnTCF1 cell lines were cultured in the presence of induction. After 24 hr and 48 hr, floating and attached cells were stained with the respective dyes. Data from both assays are presented as the mean ± standard deviation from 3 separate experiments. PI/HST assay: Cells were stained with PI (stains apoptotic/necrotic cells) and HST (stains viable cells). Percentage of PI stained cells relative to total cell number reflect the extent of cell death. Trypan blue dye exclusion assay: Cells were stained with the trypan blue dye. Percentage of trypan blue stained cells (stains dead cells) relative to total cell number reflect extent of cell death. MTT viability assay: The cells' capability to metabollically convert the MTT substrate was quantified, 48 hr after either induction (+dox) or no induction (-dox), by solubilizing MTT formazan crystals and performing spectrophotometry at 540 nm. Cell viability, which is proportional to the absorbance at 540 nm, decreases in dnTCF1 over-expressing LS174T cells relative to control cells

mRNA profiling of HCT116 cells upon ectopic expression of miR-375 and miR-375 target identification.

<p>(A) Reconstitution of mature miR-375 upon transfection with pre-miR-375 or Scr (RT-qPCR). (B) Cumulative fraction plotted as a function of log2 fold changes. The mRNAs were dichotomized according to the presence or absence of minimum one seed match in the 3′UTR or according to target prediction using Target Scan v5.2. The mRNAs with minimum one 7mer-m8 seed match within the 3′ UTR showed a higher propensity to down-regulation upon miR-375 over-expression. (C) The mRNAs were ranked according to fold change and grouped into a total of 23 bins. Upper panel: The average 7mer-8m seed frequency within the 3′UTR regions in each bin was calculated. Bottom panel: Overall miRNA-induced mRNA fold change. (D and E) Relative expression of HELLS, NOLC1, YAP1, BIRC5 and BCL2L1 upon ectopic miR-375 expression using RT-qPCR. (F) Western blots demonstrating the effect of miR-375 on the protein level of HELLS, NOLC1 and YAP1 in HCT116 cells. Loading control: β-actin. *<i>p</i><0.05. (G–H) Ago 2 immunoprecipitation. (G) RT-qPCR expression analysis of YAP1 in the cell lysates of miR-375 or Scr transfected cells (input) used for Ago2 immunoprecipitation. (H) Ago2 immunoprecipitation from cell lysates of miR-375 or Scr transfected cells (IP) followed by YAP1 expression analysis using RT-qPCR. Immunoprecipitation with a FLAG antibody was used as negative control. A 1∶1 ratio of the lysates from miR-375 and Scr transfected cells was used for FLAG immunoprecipitation. The columns represent the mean of 3 replicates ± sd.</p

Generation and characterization of stable HCT116 cells with inducible miR-375 expression (HCT116-miR-375H).

<p>(A) Dox treatment induces increased expression of miR-375 in HCT116_miR-375H cells. The Relative expression of miR-375 was measured using RT-qPCR. The columns represent the mean of 3 replicates ± sd.*p-value<0.05 when compared to HCT116_ScrH cells. (B) xCELLigence real-time monitoring of cell proliferation. The cell index from time 12–96 hours is shown. Dox was added at time 0. (C) Dox treatment significantly reduces the proliferation of HCT116_miR-375H cells. Following the real-time monitoring in B, the slope (rate of changes in cell index) was calculated from time 60–80 hours (i.e. when changes in cell viability were apparent) and presented graphically. (D) Dox treatment specifically induces Caspase 3/7 dependent apoptosis in HCT116 miR-375H cells. The Caspase 3/7 activity was examined by fluorometric kinetic analysis and expressed relative to the Caspase 3/7 activity in untreated HCT116_ScrH cells. Z-DEVD-fmk (DEVD) was added to the cells six hours post-transfection. Data are presented as ±sd. of at least 2 independent experiments each with three biological replicates. *p-value<0.05. (E) Western blotting demonstrating down-regulation of YAP1 in dox treated HCT116_miR-375H cells compared to non-treated and HCT116_ScrH cells. Loading control: β-actin. (F) miR-375 expression reduces tumor growth <i>in vivo</i>. Growth curves of tumors generated in nude mice injected with HCT116_miR-375H cells treated with (n = 4) or without (n = 4) dox in the drinking water. Dox was added to the drinking water when the tumor size was >50 mm³. Data marks and bars represent the mean ±sd. *p-value<0.05.</p

The most differentially expressed miRNAs in stage II colorectal adenocarcinomas and their ability to induce phenotypic changes in the high-throughput analysis.

1<p>A FC _(log2)≤−1.50 and ≥1.50 and a p-value≤0.01 was considered significant (Mann-Whitney U test).</p><p>NA: miRNAs not included in the pre-miRNA library from Ambion.</p><p>+: miRNAs that induced phenotypic changes (Top-40 ranked).</p><p>(+): miRNAs that induced phenotypic changes in at least one cell line (not Top-40 ranked).</p><p>A: Induction of apoptosis.</p><p>P: Inhibition of proliferation.</p

Phenotypic analyses of selected miRNAs in HCT116 cells upon ectopic expression of the miRNAs.

<p>(A) Cellular viability (MTT assay): Data are presented as ±sd. of at least 3 independent experiments each with three biological replicates and normalized to Scr. *p-value<0.05 and MTT reduction >20%. (B) Cellular death (LDH release assay): The cellular death was expressed as percentage of released LDH out of total cellular LDH. At least two independent experiments were carried out and performed in triplicates. The result of one representative experiments ±sd. is shown. *p-value<0.05. (C) Induction of apoptosis (Caspase 3/7 activity): The Caspase 3/7 activity in the lysate of pre-miRNA transfected cells was examined by fluorometric kinetic analysis and expressed relative to the Caspase 3/7 activity in “Scr” transfected cells. Data are presented as ±sd. of at least 2 independent experiments each with three biological replicates. *p-value<0.05. (D) Inhibition of miR-375 induced apoptosis by the Caspase 3/7 inhibitor z-DEVD-fmk. The Caspase 3/7 activity was measured as described in (C). Z-DEVD-fmk (DEVD) (25 µM) was added to the cells six hours post-transfection. Non treated cells (NT), Lipofectamine only (Lipo) and pre-miR miRNA Precursor Molecules-Negative Control #1 (Scr) were included as negative controls in all assays. The pre-miR-145 transfected cells were included as a positive control for performance of the MTT assay. (E) The down-regulation of miR-375 is a result of reduced expression in the epithelial cells of the tumor. Expression of miR-375 in laser capture microdissected colorectal cancer tissue. The expression was analyzed in epithelial and stromal cells from paired colorectal adenocarcinomas (n = 3) and adjacent normal (n = 3) colon mucosa LCM biopsies using RT-qPCR. The columns represent the mean expression in three samples ± sd.</p