Journal: eLife

Article Title: Differentiation of mouse fetal lung alveolar progenitors in serum-free organotypic cultures

doi: 10.7554/eLife.65811

Figure Lengend Snippet: ( A ) IC261, CX-4945, and DMAT treatments (48 h) lead to downregulation of AT2 cell markers. TTP22 and Emodin (also CK2 inhibitors) do not lead to the same phenotype, possibly due to differences in potency/target affinity. Expression levels relative to DMSO control. Three biological replicates (consisting of 2 technical replicates each) were analyzed per compound. ( B ) Casein Kinase inhibition by IC261, CX-4945, and DMAT (6 h) leads to reduced Axin2 relative expression. Treatment with TTP22 or Emodin does not significantly affect Axin2 levels. More than three biological replicates were analyzed. ( C ) CK inhibition reduced WNT/β-catenin-dependent transcription in HEK293T epithelial cells (SuperTOPFlash-based luciferase assay). ( B ) Mean values are displayed; error bars represent S.D.; p - values from one-way ANOVA, Tukey’s multiple comparisons test. ( C ) Mean values displayed; error bars represent S.D.; p-values from one-way ANOVA, Tukey’s multiple comparisons test; displayed p-values refer to comparisons with WNT3A-treated condition. Figure 4—source data 1. Normalized expression values of AT2 cell markers, CK inhibitors vs. DMSO control. Normalized Axin2 expression values, CK inhibitors vs. DMSO control and related statistics. Raw luciferase data. Normalized luciferase values and related statistics.

Article Snippet: Transfected cells were serum-starved for 6 h and treated with 500 ng/ml human recombinant WNT3A (Proteintech HZ-1296) and 10 μM chemical compounds or 0.1% DMSO for 12 h. Cells were washed with PBS on ice to remove phenol red and lysed.

Techniques: Expressing, Control, Inhibition, Luciferase

Journal: eLife

Article Title: Differentiation of mouse fetal lung alveolar progenitors in serum-free organotypic cultures

doi: 10.7554/eLife.65811

Figure Lengend Snippet:

Article Snippet: Transfected cells were serum-starved for 6 h and treated with 500 ng/ml human recombinant WNT3A (Proteintech HZ-1296) and 10 μM chemical compounds or 0.1% DMSO for 12 h. Cells were washed with PBS on ice to remove phenol red and lysed.

Techniques: Recombinant, Transfection, Construct, Plasmid Preparation, Mutagenesis, Reporter Assay, Reverse Transcription, SYBR Green Assay, Sequencing, Software

Journal: Cell

Article Title: Vitamin D switches BAF complexes to protect β cells

doi: 10.1016/j.cell.2018.04.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS Differentiation of hiPSC to human β-like cells As previously described ( Yoshihara et al., 2016 ), INS-GFP hiPSCs (derived from HUVECs) were cultured to 90% confluency in complete mTeSR Media, at which time the media was changed to 100 ng/ml Activin (R&D Systems, 338-AC), 25 ng/ml Wnt3a (R&D Systems, 5036-WN) in differentiation media (800 ml DMEM/F12, 13.28 g BSA, 10 ml Glutamax, 560 mg NaHCO3, 330 mg thiamine, 100 mg reduced glutathione, 3300 mg vitamin C, 14 μg selenium, 10 ml NEAA, 2 ml trace element B, 1 ml trace element C, 7 μl β-ME, 2 ml DLC, 2 ml GABA, 2 ml LiCl, 129.7 μg pipecolic acid, insulin 2 mg up to 1,000 ml) for 2 days and then 100 ng/ml activin in differentiation media for another 2 days.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: Cells

Article Title: LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer

doi: 10.3390/cells12222658

Figure Lengend Snippet: Expression of WNT5A and LGR5 proteins in colon cancer tissue and their relation to the survival of these patients. ( A ) Representative IHC staining images of the WNT5A protein in normal mucosa and matched colon cancer tissue and their mean IRS values are presented in the graph. ( B ) Representative IHC staining images of the LGR5 protein in normal mucosa and matched colon cancer tissue and their mean IRS values are presented in the graph. ( C ) Representative IHC staining images of low and high WNT5A protein expression in colon cancer tissue. ( D ) Multivariate overall five-year survival analysis for low-risk (high WNT5A expression) and high-risk (low WNT5A expression) patients when adjusted for sex, LNM and TNM stage . ( E ) Representative IHC staining images of low and high LGR5 protein expression in colon cancer tissue. ( F ) Multivariate overall five-year survival analysis for low-risk (low LGR5 expression) and high-risk (high LGR5 expression) patients when adjusted for the prognostic factors sex and TNM stage . The results in Panels A and B are shown as the mean ± SEM; *** p < 0.001, analyzed with Wilcoxon’s match paired Student’s t test.

Article Snippet: Fifty-seven patient tumor samples were available for the evaluation of WNT5A expression, and these samples were analyzed with the anti-WNT5A antibody (AF645, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemistry

Journal: Cells

Article Title: LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer

doi: 10.3390/cells12222658

Figure Lengend Snippet: mRNA expression levels of WNT5A and LGR5 in colon cancer tissue and their correlations with the survival of colon cancer patients based on data from publicly available cohorts. Using the TCGA-COAD cohort, we performed univariate five-year overall survival analysis for ( A ) WNT5A and ( B ) LGR5. ( C ) Five-year overall survival analysis of 4 possible groups based on the combinations of WNT5A (low and high) and LGR5 (low and high) mRNA expressions. ( D ) Spearman’s correlation test between WNT5A and LGR5 mRNA expressions using the publicly available GSE44076 cohort.

Article Snippet: Fifty-seven patient tumor samples were available for the evaluation of WNT5A expression, and these samples were analyzed with the anti-WNT5A antibody (AF645, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Expressing

Journal: Cells

Article Title: LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer

doi: 10.3390/cells12222658

Figure Lengend Snippet: Effects of Foxy5 on LGR5 and β-catenin in colon cancer cells and correlation between β-catenin and WNT5A transcript expressions. SW480 colon cancer cells were treated with either vehicle or 100 µM Foxy5 for 24 h. ( A ) Representative immunofluorescence images of LGR5 and active β-catenin in SW480 cells treated with vehicle (left panels) or Foxy5 (right panels). The scale bars represent 10 µm in the upper panel and 5 µm in the lower panel. The mean fluorescence intensity (MFI) of ( B ) LGR5, ( C ) total active β-catenin, and ( D ) nuclear active β-catenin are outlined in their respective graphs and shown as the mean ± SEM for at least three independent experiments. * p < 0.05 and ** p < 0.01, analyzed with Mann-Whitney unpaired Student’s t test. ( E ) Spearman’s correlation test reveals a correlation between the expression of CTNNB1 and WNT5A transcripts based on data from the GSE44076 cohort.

Article Snippet: Fifty-seven patient tumor samples were available for the evaluation of WNT5A expression, and these samples were analyzed with the anti-WNT5A antibody (AF645, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Immunofluorescence, Fluorescence, MANN-WHITNEY, Expressing

Journal: Cells

Article Title: LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer

doi: 10.3390/cells12222658

Figure Lengend Snippet: The expression of the LGR5 ligand RSPO3 correlates positively with LGR5 expression and negatively with WNT5A expression in colon cancer tissue and is downregulated by Foxy5. ( A ) Representative IHC staining images of the RSPO3 protein in normal mucosa and matched colon cancer tissue and their mean IRS values are presented in the graph. The results are shown as the mean ± SEM; * p < 0.05, analyzed with Wilcoxon match-paired Student’s t test. ( B ) Spearman’s correlation test between LGR5 and RSPO3 protein expressions in the Malmö-CC cohort. ( C ) Spearman’s correlation test between LGR5 and RSPO3 mRNA expressions using the publicly available GSE44076 cohort. ( D ) Spearman’s correlation test between RSPO3 and WNT5A mRNA expressions using the publicly available GSE44076 cohort. ( E ) RSPO3 mRNA expression in HT-29 colon cancer cells stimulated in vitro with or without 100 µM Foxy5 for 24 h. The result in the graph is shown as the mean ± SEM for at least three independent experiments. ( F ) RSPO3 mRNA levels in HT-29 colon cancer tissues from xenograft mice treated with vehicle or Foxy5 (2 µg/g) every other day for a 14-day period. The results in the graph are shown as the mean ± SEM. ( G ) Representative IHC images of RSPO3 protein expression in the HT-29 cell xenografts from mice treated with vehicle or Foxy5 (2 µg/g) every other day for a 14-day period. The results in Panels ( F , G ) are presented in the graph as the mean ± SEM, and 5 xenograft tissues from 5 different mice were analyzed per group of animals. * p < 0.05, ** p < 0.01, analyzed with Mann-Whitney unpaired Student’s t test.

Article Snippet: Fifty-seven patient tumor samples were available for the evaluation of WNT5A expression, and these samples were analyzed with the anti-WNT5A antibody (AF645, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemistry, In Vitro, MANN-WHITNEY

Journal: Cells

Article Title: LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer

doi: 10.3390/cells12222658

Figure Lengend Snippet: The expression of the β-catenin target VEGFA predicts a poor prognosis, correlates with the expression of WNT5A and β-catenin at the transcript level in colon cancer tissue, and is downregulated by WNT5A signaling. ( A ) Using the TCGA-COAD cohort, we performed univariate five-year overall survival analysis for VEGFA. ( B ) Five-year overall survival analysis of 4 possible patient groups from the TCGA-COAD cohort based on the combinations of VEGFA (low and high) and WNT5A (low and high) mRNA expressions. Spearman’s correlation tests are shown between ( C ) VEGFA and WNT5A mRNA expression levels and ( D ) VEGFA and β-catenin mRNA (CTNNB1). Both correlations were made based on data from the publicly available GSE44076 cohort. ( E ) This panel outlines VEGFA mRNA expression in HT-29 colon cancer cells stimulated in vitro with or without 400 ng/mL rWNT5A or 100 µM Foxy5 for 24 h. ( F ) Representative Western blot and the accumulated densitometric VEGFA protein expression in HCT-116 colon cancer cells stimulated in vitro with or without 400 ng/mL rWNT5A or 100 µM Foxy5 for 24 h. The results in the graphs are shown as the mean ± SEM for at least three independent experiments. ( G ) VEGFA mRNA levels in HT-29 colon cancer tissues from xenograft mice treated with vehicle or Foxy5 (2 µg/g) every other day for a 14-day period. ( H ) Representative IHC images of VEGFA protein expression in the HT-29 colon cancer cell xenografts from mice treated with vehicle or Foxy5 (2 µg/g) every other day for a 14-day period. The results in panels ( G , H ) are presented in the graphs as the mean ± SEM (5 xenograft tissues from 5 different mice were analyzed per group of animals); * p < 0.05, ** p < 0.01 and *** p < 0.001, analyzed with Mann-Whitney unpaired Student’s t test.

Article Snippet: Fifty-seven patient tumor samples were available for the evaluation of WNT5A expression, and these samples were analyzed with the anti-WNT5A antibody (AF645, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Expressing, In Vitro, Western Blot, MANN-WHITNEY

Journal: Cells

Article Title: LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer

doi: 10.3390/cells12222658

Figure Lengend Snippet: The expression of the colon cancer stem cell marker DCLK1 predicts a poor prognosis, correlates with the expression of WNT5A in colon cancer tissue, and is downregulated by Foxy5. ( A ) Representative IHC staining images of the DCLK1 protein in normal mucosa and matched colon cancer tissue and their mean IRS values are presented in the graph. The results are given as the mean ± SEM; *** p < 0.001, analyzed with Wilcoxon match-paired Student’s t test. ( B ) Representative IHC staining images of low and high DCLK1 protein expression in colon cancer tissue. ( C ) Univariate five-year overall survival analysis of patients with either low or high DCLK1 expression in their tumors. ( D ) Multivariate five-year overall survival analysis for low-risk (low DCLK1 expression) and high-risk (high DCLK1 expression) patients when adjusted for sex, LNM and TNM stage . ( E ) Using the TCGA-COAD cohort we performed univariate five-year overall survival analysis for DCLK1. ( F ) Five-year overall survival analysis of four possible groups from the TCGA-COAD cohort based on the combinations of DCLK1 (low and high) and WNT5A (low and high) mRNA expressions. ( G ) Spearman’s correlation test between DCLK1 and WNT5A mRNA expressions based on data from the publicly available GSE44076 cohort. ( H ) HCT-116 colon cancer cells were grown to form colonospheres, and then the cells were treated with either vehicle or 100 µM Foxy5. ( I ) DCLK1 mRNA expression in HT-29 colon cancer cells stimulated in vitro with or without 100 µM Foxy5. The effects of Foxy5 treatment on DCLK1 expression were analyzed by Western blotting (panel H ) or RT-qPCR (panel I ) and the results are presented in the graphs as the mean ± SEM for at least three independent experiments; ** p < 0.01, analyzed with Mann-Whitney unpaired Student’s t test.

Article Snippet: Fifty-seven patient tumor samples were available for the evaluation of WNT5A expression, and these samples were analyzed with the anti-WNT5A antibody (AF645, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Expressing, Marker, Immunohistochemistry, In Vitro, Western Blot, Quantitative RT-PCR, MANN-WHITNEY