Wnt/beta-catenin

Fluorescence microscopy of pSuper or Cdo shRNA expressing P19 cells at ITS1 immunostained withβ-tubulin III antibodies. Size bar=100 um. P19/control or P19/Cdo shRNA cells were treated with DMSO or XAV939 in the differentiation medium for 72 h followed by immunostaining.

ICG-001 variably influences Wnt transcriptional activity across pancreatic cancer lines. Nuclear extracts from AsPC-1 cells treated with vehicle or 30 umol/L ICG-001 for 6 hours were immunoprecipitated with anti-CBP or control IgG antibodies followed by Western blot analyses for β-catenin and CBP.

(a) MET inhibition by INCB28060 prevents HGF-induced MET phosphorylation in MDA-MB231 and HCC-1954 cells. After one hour preincubation with INC2B8060, BC cells were stimulated with HGF for one hour. Phosphorylation of MET and total MET were determined by western blot and quantified by densitometric analysis. Results are expressed as percentage of unstimulated and untreated cells (negative control).

Effect of IWR-1 treatment on total β-catenin levels and adipogenesis in high-passage shSCR and shTCF7L2 cells. Cells were treated with IWR-1 (10 μmol/L) every other day for 6 days during differentiation. Western blot demonstrates the effect of IWR-1 on total β-catenin protein levels at the end of adipogenesis (each lane represents a single independent adipogenesis experiment, n = 4 for each treatment).

Transient Wnt inhibition results in persistent decrease of b-catenin. HTM cells were incubated for 4 days in serum-free media, treated with Wnt inhibitors for 8 days in serum free media, and recovered for 4 days in serum-free media before being fixed and stained for b-catenin. (B) HTM cells treated with 10 μM KY02111 or LGK-974 likewise show a reduction of b-catenin staining intensity. (CeD) Quantification of b-catenin staining intensity in HTM cells from 3 donors indicates a statistically significant decrease in intensity after 0.5 μg/mL SFRP1 (SFRP1), 10 μM KY02111, and 10 μM LGK-974. (E) Western blot shows increased T41 phosphorylation of b-catenin after SFRP1 or KY02111 treatment. Data presented as mean ± SEM. Significant difference from control using Tukey's method denoted by * p < 0.05; ***p < 0.001; using Student's T-test denoted by #p < 0.05. n=3. Scale bar 50 μm.

KIF3A knockdown promotes both ligand secretion and intracellular signalling of the Wnt/β-catenin pathway in NSCLC cell lines. (c,d) SuperTOPFlash reporter assay to examine the effect of KIF3A knockdown on signalling pathway activation by autocrine or exogenous WNTs in A549 and SW900 cell lines. A549 and SW900 cells were incubated with DMSO or IWP-2 (30 μ M) for 48 h, and then treated with BSA or WNT3A (100 ng/ml) for additional 24 h. Luciferase reporter activity was calculated by dividing TOP/RL ratio by FOP/RL ratio (n = 3; error bars indicate SEM; p-value was determined by one-way ANOVA). *P < 0.05; **P < 0.01.

The salinomycin inhibited the growth of CNE-2 in time- and dose-dependent manner. SAL salinomycin

Ki-67 staining in xenograft tissues from FH535-treated and control group. Magnifications: ×200 in left column, ×400 in right column.

A549 cells were treated with KYA1797K for 72 h, cells lysates were collected for western blotting, concentration gradient (1, 12.5 μM, 25 μM) was set for the analysis

Inhibition of GSK3 activity induces an autophagic response in human pancreatic cancer cells. PANC1 cells were treated for 24 h with DMSO, CHIR99021 (CHIR; 5 uM) or the mTOR inhibitor Torin1 (250 nM). Autophagosome (punctate LC3B) (D) and lysosome (E) labeling was visualized with a Zeiss LSM700 confocal microscope. Images were acquired with a 20x (D) or 63x (E) objective. Nuclei were stained with DAPI. Scale bars, 20 祄.

For MTT assays, cells (2,000 ~ 5,000 cells/well) were subcultured into 96-well plates according to their growth properties. Cell proliferation was assayed at 72 hr after treatment of CHIR-99021 by adding 20 μl of 5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution per 100 μl of growth medium. After incubating for 3-4 h at 37°C, the media were removed and 150 µl/well of MTT solvent (either absolute DMSO or isopropanol containing 4 mM HCl and 0.1% Nonidet-40) was added to dissolve the formazan. The absorbance of each well was measured by ELx808 (BioTek, Winooski, VT) or Wallac Victor2 (Perkin-Elmer Life Sciences, Boston, MA) Microplate Reader. Viable cells are presented as percent of control, vehicle-treated cells. 

PC-3 cells were seeded in 6-well plates. On the next day, cells were treated with the indicated concentrations of 3-AWA, LY294002, CGP57380 or Rapamycin alone and or combination for 12 h. The cells were then subjected to preparation of whole-cell lysates for detection of the indicated proteins using western blotting. The western blots are the representative of three similar experiments.

Both Wnt sinaling activators of agonist and recombinant protein, and therapeutic agents of fluorouracil and docetaxel stimulated TCF-4 promoter functions of wild-type (wt) could be blocked by Nano-Let-7b, compared with negative control group of mutant-type (mut). In detail, Let-7b alone affected the TCF-4 promoter activity effectively, and wnt signaling activators of either Wnt agonist-1 or Wnt protein attenuated the Let-7b functions significantly. Combined usage of therapeutic agent of either fluorouracil or docetaxel with Let-7b increased the TCF-4 activity when compared to groups treated with Let-7b alone, however, the TCF-4 activity when applying combination was still significantly lower than that of controlled group

Western blots of nuclear and cytoplasmic fractions of β-catenin and total CDH3. d The ratio of nuclear/cytosolic of β-catenin was calculated by densitometry analysis.

Fluorescence microscopy of pSuper or Cdo shRNA expressing P19 cells at ITS1 immunostained withβ-tubulin III antibodies. Size bar=100 um. P19/control or P19/Cdo shRNA cells were treated with DMSO or XAV939 in the differentiation medium for 72 h followed by immunostaining.

ICG-001 variably influences Wnt transcriptional activity across pancreatic cancer lines. Nuclear extracts from AsPC-1 cells treated with vehicle or 30 umol/L ICG-001 for 6 hours were immunoprecipitated with anti-CBP or control IgG antibodies followed by Western blot analyses for β-catenin and CBP.

(a) MET inhibition by INCB28060 prevents HGF-induced MET phosphorylation in MDA-MB231 and HCC-1954 cells. After one hour preincubation with INC2B8060, BC cells were stimulated with HGF for one hour. Phosphorylation of MET and total MET were determined by western blot and quantified by densitometric analysis. Results are expressed as percentage of unstimulated and untreated cells (negative control).

Effect of IWR-1 treatment on total β-catenin levels and adipogenesis in high-passage shSCR and shTCF7L2 cells. Cells were treated with IWR-1 (10 μmol/L) every other day for 6 days during differentiation. Western blot demonstrates the effect of IWR-1 on total β-catenin protein levels at the end of adipogenesis (each lane represents a single independent adipogenesis experiment, n = 4 for each treatment).

Transient Wnt inhibition results in persistent decrease of b-catenin. HTM cells were incubated for 4 days in serum-free media, treated with Wnt inhibitors for 8 days in serum free media, and recovered for 4 days in serum-free media before being fixed and stained for b-catenin. (B) HTM cells treated with 10 μM KY02111 or LGK-974 likewise show a reduction of b-catenin staining intensity. (CeD) Quantification of b-catenin staining intensity in HTM cells from 3 donors indicates a statistically significant decrease in intensity after 0.5 μg/mL SFRP1 (SFRP1), 10 μM KY02111, and 10 μM LGK-974. (E) Western blot shows increased T41 phosphorylation of b-catenin after SFRP1 or KY02111 treatment. Data presented as mean ± SEM. Significant difference from control using Tukey's method denoted by * p < 0.05; ***p < 0.001; using Student's T-test denoted by #p < 0.05. n=3. Scale bar 50 μm.

KIF3A knockdown promotes both ligand secretion and intracellular signalling of the Wnt/β-catenin pathway in NSCLC cell lines. (c,d) SuperTOPFlash reporter assay to examine the effect of KIF3A knockdown on signalling pathway activation by autocrine or exogenous WNTs in A549 and SW900 cell lines. A549 and SW900 cells were incubated with DMSO or IWP-2 (30 μ M) for 48 h, and then treated with BSA or WNT3A (100 ng/ml) for additional 24 h. Luciferase reporter activity was calculated by dividing TOP/RL ratio by FOP/RL ratio (n = 3; error bars indicate SEM; p-value was determined by one-way ANOVA). *P < 0.05; **P < 0.01.

The salinomycin inhibited the growth of CNE-2 in time- and dose-dependent manner. SAL salinomycin

Ki-67 staining in xenograft tissues from FH535-treated and control group. Magnifications: ×200 in left column, ×400 in right column.

A549 cells were treated with KYA1797K for 72 h, cells lysates were collected for western blotting, concentration gradient (1, 12.5 μM, 25 μM) was set for the analysis

Inhibition of GSK3 activity induces an autophagic response in human pancreatic cancer cells. PANC1 cells were treated for 24 h with DMSO, CHIR99021 (CHIR; 5 uM) or the mTOR inhibitor Torin1 (250 nM). Autophagosome (punctate LC3B) (D) and lysosome (E) labeling was visualized with a Zeiss LSM700 confocal microscope. Images were acquired with a 20x (D) or 63x (E) objective. Nuclei were stained with DAPI. Scale bars, 20 祄.

For MTT assays, cells (2,000 ~ 5,000 cells/well) were subcultured into 96-well plates according to their growth properties. Cell proliferation was assayed at 72 hr after treatment of CHIR-99021 by adding 20 μl of 5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution per 100 μl of growth medium. After incubating for 3-4 h at 37°C, the media were removed and 150 µl/well of MTT solvent (either absolute DMSO or isopropanol containing 4 mM HCl and 0.1% Nonidet-40) was added to dissolve the formazan. The absorbance of each well was measured by ELx808 (BioTek, Winooski, VT) or Wallac Victor2 (Perkin-Elmer Life Sciences, Boston, MA) Microplate Reader. Viable cells are presented as percent of control, vehicle-treated cells. 

PC-3 cells were seeded in 6-well plates. On the next day, cells were treated with the indicated concentrations of 3-AWA, LY294002, CGP57380 or Rapamycin alone and or combination for 12 h. The cells were then subjected to preparation of whole-cell lysates for detection of the indicated proteins using western blotting. The western blots are the representative of three similar experiments.

Both Wnt sinaling activators of agonist and recombinant protein, and therapeutic agents of fluorouracil and docetaxel stimulated TCF-4 promoter functions of wild-type (wt) could be blocked by Nano-Let-7b, compared with negative control group of mutant-type (mut). In detail, Let-7b alone affected the TCF-4 promoter activity effectively, and wnt signaling activators of either Wnt agonist-1 or Wnt protein attenuated the Let-7b functions significantly. Combined usage of therapeutic agent of either fluorouracil or docetaxel with Let-7b increased the TCF-4 activity when compared to groups treated with Let-7b alone, however, the TCF-4 activity when applying combination was still significantly lower than that of controlled group

Western blots of nuclear and cytoplasmic fractions of β-catenin and total CDH3. d The ratio of nuclear/cytosolic of β-catenin was calculated by densitometry analysis.