Journal: Molecular Oncology

Article Title: TRPM 4 regulates Akt/ GSK 3‐β activity and enhances β‐catenin signaling and cell proliferation in prostate cancer cells

doi: 10.1002/1878-0261.12100

Figure Lengend Snippet: TRPM 4 expression affects protein levels and phosphorylation status of β‐catenin. (A) mRNA expression of TRPM 4 gene in PC 3 WT and LNC aP WT cells relative to nontumoral RWPE ‐1 prostate cells. RT ‐ qPCR were performed at least three times. T‐ test with Welch correction, ns P > 0.05, ** P ≤ 0.01, *** P ≤ 0.001. (B) Western blot was performed to detect TRPM 4 expression in prostate cancer cell lines. Representative western blots and their densitometries from three independent experiments. Mean ± SEM . T‐ test with Welch correction, * P ≤ 0.05. (C) Knockdown of TRPM 4 in PC 3 cells resulted in a significant decrease in total β‐catenin protein levels, along with the nonphosphorylated β‐catenin (‘active’), and an increase in phosphorylated β‐catenin at Ser33/37/Thr41 residues. T‐ test with Welch correction, ** P ≤ 0.01. (D, E) TRPM 4‐transfected LNC aP cells show an increase in total and nonphosphorylated β‐catenin protein levels compared to MOCK ‐transfected cells. T‐ test with Welch correction, * P ≤ 0.05, ** P ≤ 0.01. Representative western blots and their densitometries from at least three experiments. Mean ± SEM are shown.

Article Snippet: Cells were transiently transfected with 0.5 μg of constitutively active vector encoding Renilla luciferase (Promega, Madison, WI, USA) and 2 μg of β‐catenin responsive firefly luciferase reporter plasmid TopFlash or the negative control FopFlash (Merck Millipore, Billerica, MA, USA).

Techniques: Expressing, Phospho-proteomics, Quantitative RT-PCR, Western Blot, Knockdown, Transfection

Journal: Molecular Oncology

Article Title: TRPM 4 regulates Akt/ GSK 3‐β activity and enhances β‐catenin signaling and cell proliferation in prostate cancer cells

doi: 10.1002/1878-0261.12100

Figure Lengend Snippet: TRPM 4 expression effects, nuclear distribution, and transcriptional activity of β‐catenin. (A, B) Knockdown of TRPM 4 in PC 3 cells decreases β‐catenin nuclear localization. Representative images of intracellular β‐catenin detected by immunofluorescence in PC 3 ShControl (upper) and PC 3 Sh TRPM 4 (bottom). Scale bar 20 μ m . Graph shows arbitrary units of fluorescence of β‐catenin nuclear/cytoplasmic ratio quantified in at least three independent experiments. Mean ± SEM are shown. T‐ test with Welch correction, *** P ≤ 0.001. (C) Knockdown of TRPM 4 in PC 3 cells decreases β‐catenin cotranscriptional activity. PC 3 Sh TRPM 4 and PC 3 ShControl cells were transfected with plasmid pTOP or pFOP and pTK ‐Renilla as normalizer. Normalized TOP / FOP Luciferase activity is shown. T‐ test with Welch correction, *** P ≤ 0.001. (D, E) Relative mRNA expression of β‐catenin target genes. Axin2, survivin, c‐Myc, and cyclin D1 genes were analyzed in PC 3 Sh TRPM 4 compared to PC 3 ShControl cells with RT ‐ qPCR assay. All experiments were performed at least three times. Mean ± SEM are shown . T‐ test with Welch correction, *** P ≤ 0.001.

Article Snippet: Cells were transiently transfected with 0.5 μg of constitutively active vector encoding Renilla luciferase (Promega, Madison, WI, USA) and 2 μg of β‐catenin responsive firefly luciferase reporter plasmid TopFlash or the negative control FopFlash (Merck Millipore, Billerica, MA, USA).

Techniques: Expressing, Activity Assay, Knockdown, Immunofluorescence, Fluorescence, Transfection, Plasmid Preparation, Luciferase, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: TRPM 4 regulates Akt/ GSK 3‐β activity and enhances β‐catenin signaling and cell proliferation in prostate cancer cells

doi: 10.1002/1878-0261.12100

Figure Lengend Snippet: Activity of TRPM 4 channel is required for β‐catenin stability and GSK ‐3β inhibitory phosphorylation. Overexpression of TRPM 4 in T‐ RE x 293 ( TREX ) cells was induced by previous incubation with tetracycline 1 μg·mL −1 (Tet). Cells were incubated for 2 h with the specific TRPM 4 inhibitor 9‐phenanthrol at 10 μ m or DMSO (vehicle) before protein extraction. (A, B) TRPM 4 overexpression enhances β‐catenin protein levels, decreases its inhibitory phosphorylation, and increases the GSK ‐3β S9 phosphorylation. The presence of 9‐phenanthrol reverts the effects of the TRPM 4 overexpression on these intracellular proteins. (A) Representative western blots and the densitometries of at least three independent experiments (mean ± SEM ) are shown. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, multiple t ‐test comparisons using t ‐test with Welch correction.

Article Snippet: Cells were transiently transfected with 0.5 μg of constitutively active vector encoding Renilla luciferase (Promega, Madison, WI, USA) and 2 μg of β‐catenin responsive firefly luciferase reporter plasmid TopFlash or the negative control FopFlash (Merck Millipore, Billerica, MA, USA).

Techniques: Activity Assay, Phospho-proteomics, Over Expression, Incubation, Protein Extraction, Western Blot

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA lncARSR promotes epithelial ovarian cancer cell proliferation and invasion by association with HuR and miR-200 family

doi:

Figure Lengend Snippet: lncARSR activates Wnt/β-catenin signaling pathway in EOC cells. A. Luciferase activity of TOPflash reporter was evaluated in SKOV3 cells overexpressing lncARSR. B. Luciferase activity of TOPflash reporter was evaluated in CAOV3 cells with lncARSR knockdown. C. The mRNA and protein level of β-catenin was determined by qRT-PCR and western blot in SKOV3 cells overexpressing lncARSR, respectively. D. The mRNA and protein level of β-catenin was determined by qRT-PCR and western blot in CAOV3 cells with lncARSR knockdown, respectively. E. The correlation between lncARSR and β-catenin mRNA expression was determined in EOC tissue samples. F. The mRNA and protein level of c-myc and cyclin D1 was determined by qRT-PCR and western blot in SKOV3 cells overexpressing lncARSR, respectively. G. The mRNA and protein level of c-myc and cyclin D1 was determined by qRT-PCR and western blot in CAOV3 cells with lncARSR knockdown, respectively. Data are shown as means ± SEM, *P < 0.05.

Article Snippet: β-catenin/TCF firefly luciferase reporter construct (TOPflash) and pRL-TK reporter was purchased from Promega (Madison, WI, USA).

Techniques: Luciferase, Activity Assay, Knockdown, Quantitative RT-PCR, Western Blot, Expressing

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA lncARSR promotes epithelial ovarian cancer cell proliferation and invasion by association with HuR and miR-200 family

doi:

Figure Lengend Snippet: lncARSR interacts with miR-200 family. A. MS2-RIP followed by microRNA qRT-PCR to detect microRNAs endogenously associated with lncARSR. B. Luciferase activity in EOC cells co-transfected with miR-200s and luciferase reporters containing nothing, lncARSR or mutant transcript. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. C. The relative expression of lncARSR in EOC cells transiently overexpressing miR-200s. D. Anti-AGO2 RIP was performed in EOC cells transiently overexpressing miR-200s, followed by qRT-PCR to detect lncARSR associated with AGO2. E. The relative expression of miR-200s in EOC cells transiently overexpressing lncARSR or lncARSR-mut. Data are shown as means ± SEM, *P < 0.05.

Article Snippet: β-catenin/TCF firefly luciferase reporter construct (TOPflash) and pRL-TK reporter was purchased from Promega (Madison, WI, USA).

Techniques: Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: Henryin, an ent-kaurane Diterpenoid, Inhibits Wnt Signaling through Interference with β-Catenin/TCF4 Interaction in Colorectal Cancer Cells

doi: 10.1371/journal.pone.0068525

Figure Lengend Snippet: (A) Henryin does not affect the amount of total β-catenin and pho-β-catenin in SW480 cells. (B) Henryin does not affect the distribution of β-catenin in cytosol and nucleus fractions in SW480 cells. SW480 cells were treated with the indicated dosages of henryin for 24h. The cell extracts or fractions were prepared and analyzed by western blotting. Lamin A/C and β-actin were used as loading controls of nuclei and cytoplasm proteins, respectively. (C) Henryin antagonizes the Wnt signaling stimulated with LiCl or β-catenin. HEK293T cells were transiently transfected with ST-Luc and β-catenin or constitutively active β-catenin (S37A) plasmids, respectively, or treated with LiCl (20mM). Around 3h after transfection or treatment, henryin was added and cells were incubated for an additional 24h. Each bar is the mean ± SD from three independent experiments. (D) Henryin, but not enmenol and minheryin C, reduced the TCF4 levels associated with β-catenin in SW480 cells in a dose dependent manner. Cells were treated with the indicated dosages of henryin and its analogs enmenol and minheryin C for 12h and immune-precipitation was performed by β-catenin antibody with mouse IgG as a control, and then TCF4 was detected by western blotting. (E) Henryin directly disrupts the interaction of β-catenin with TCF4 in the in vitro binding assay. Human recombinant β-catenin (0.8µg) and TCF4 (0.5µg) were mixed, incubated with different concentrations of henryin and its analogs enmenol and minheryin C, and the mixture was subjected to co-immunoprecipitation with β-catenin antibody and western blotting analysis with TCF4 antibody, with mouse IgG used as control. (F) Quantification of the western blots shown in D and E. The software ImageJ was used to analyze the intensities of bands. Data was presented as mean ± SD from three experiments. Hen, henryin, En, enmenol, Min, minheryin C.

Article Snippet: SW480 and HCT116 cells were co-transfected with 100ng of plasmids in total, including 80ng of a well-characterized Wnt/β-catenin pathway responsive firefly luciferase reporter plasmid SuperTOPflash (ST-Luc) and 20ng of pRL-SV40 (Promega) control reporter plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Incubation, In Vitro, Binding Assay, Recombinant, Immunoprecipitation, Software