Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Lack of stromal cell activation 24 hours after exposure to 12 Gy TBI upon CPN treatment. ( A ) RT-qPCR analysis of Gli1 in stromal cells isolated from Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 24 hours pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. ( B ) Representative images of Western blot analysis of GLI1 in stromal cells obtained from Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 24 hours pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Actin was used as a loading control. ( C ) Quantification of GLI1 levels in stromal cells obtained from Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 24 hours pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. ( D ) Representative images of immunofluorescence staining for Bmi1-Cre ER -positive cells marker (YFP), porcupine enzyme (PORCN), proliferation marker (EdU), and nuclei marker (DAPI) in the duodenum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 24 hours pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Arrows indicate PORCN- or EdU-positive cells, respectively. Scale bar = 50 μm. ( E ) Quantification of PORCN-positive cells per DAPI-positive cells among 200-crypts-long fragments of the duodenum from mice exposed to 0 or 12 Gy TBI at 24 hours pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. ( F ) Quantification of EdU-positive cells per DAPI-positive cells among 200-crypts-long fragments of the duodenum from mice exposed to 0 or 12 Gy TBI at 24 hours pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. ( G–J ) RT-qPCR analysis of WNT ligand encoding genes’ expression: ( G ) Wnt2b , ( H ) Wnt4 , ( I ) Wnt5a , and ( J ) Rspo3 in stromal cells isolated from Bmi1-Cre ER mice pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN, and exposed to 0 or 12 Gy TBI at 24 hours. Data points represent the average of 5 independent experiments with the mean ± SD indicated. Significance was determined by 1-way analysis of variance followed by an analysis of the normal distribution (Tukey’s test). ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Activation Assay, Quantitative RT-PCR, Isolation, Western Blot, Control, Immunofluorescence, Staining, Marker, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Time-course analysis of PORCN and EdU expression upon CPN treatment. Representative images of immunofluorescence staining for Bmi1Cre ER -positive cells marker (YFP), porcupine enzyme (PORCN), proliferation marker (EdU), and nuclei marker (DAPI) in the duodenum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 0, 24, 48, 72, and 96 hours, and pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Arrows indicate PORCN- or EdU-positive cells, respectively. Scale bar = 50 μm.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Expressing, Immunofluorescence, Staining, Marker

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Time-course analysis of SHH expression upon CPN treatment. Representative images of immunofluorescence staining for Bmi1-Cre ER positive cells marker (YFP), SHH, proliferation marker (EdU), and nuclei marker (DAPI) in the duodenum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 0, 24, 48, 72, and 96 hours, and pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Scale bar = 100 μm.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Expressing, Immunofluorescence, Staining, Marker

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Determination of the effective dose of mouse recombinant SHH-N and CPN influencing stromal cell activity in vitro. ( A , B ) Dose-dependent RT-qPCR analysis of Gli1 in primary cultured stromal cells isolated from the Bmi1-Cre ER mice incubated with different doses of ( A ) mouse rSHH-N or vehicle, BSA, or ( B ) CPN or vehicle, ethanol. Fold change was calculated vs control cells maintained in ADF medium. Data points represent the average of 3 independent experiments with the mean ± SD indicated. ( C ) Representative merged (bright field and GFP channel) images of Bmi1-Cre ER –derived organoid regeneration on day 8, cultured with stromal cells obtained from non-irradiated or irradiated-source with or without BSA or ethanol. Scale bar = 50 μm. ( D ) Quantification of the area of regenerating organoids. Data points represent the average of 3–5 independent experiments with the mean ± SD indicated. Significance was determined by the Student’s test for panels A and B and 1-way analysis of variance for panel D followed by an analysis of the normal distribution (Tukey’s test). ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Recombinant, Activity Assay, In Vitro, Quantitative RT-PCR, Cell Culture, Isolation, Incubation, Control, Derivative Assay, Irradiation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Inhibition of the SHH pathway impairs intestinal epithelium regeneration and reduces MSI1 expression in vivo. ( A ) Representative images of hematoxylin and eosin staining in the duodenum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 96 hours and pretreated with tamoxifen (TAM), TAM and vehicle (TAM + HBC), or TAM and SHH signaling inhibitor, CPN (TAM + CPN). Scale bar = 100 μm. ( B ) Representative images of immunofluorescence staining for Bmi1 YFP –positive cells marker (YFP), proliferation marker (EdU), MSI1, and nuclei marker (DAPI) in the duodenum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 96 hours and pretreated with TAM, TAM and vehicle (TAM + HBC), or TAM and SHH signaling inhibitor, CPM (TAM + CPN). Scale bar = 100 μm. ( C ) Quantification of regenerating YFP-positive crypts among 200-crypts-long fragments of the duodenum from mice exposed to 12 Gy TBI at 96 hours. ( D ) Quantification of regenerating YFP-positive crypts among all YFP-positive crypts among 200-crypts-long fragments of the duodenum from mice exposed to 12 Gy TBI at 96 hours. Data points represent the average of 5 independent experiments with the mean ± SD indicated. Significance was determined by the Student’s test followed by an analysis of the normal distribution (Tukey’s test). ∗∗∗ P < .001; ∗∗∗∗ P < .001.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Inhibition, Expressing, In Vivo, Staining, Immunofluorescence, Marker

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Inhibition of the SHH pathway impairs intestinal epithelium regeneration in duodenum. Representative images of hematoxylin and eosin staining of the duodenum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 0, 24, 48, 72, and 96 hours and pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Scale bar = 100 μm.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Inhibition, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Inhibition of the SHH pathway impairs intestinal epithelium regeneration in jejunum. Representative images of hematoxylin and eosin staining of the jejunum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 0, 24, 48, 72, and 96 hours and pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Scale bar = 100 μm.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Inhibition, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Inhibition of the SHH pathway impairs intestinal epithelium regeneration in ileum. Representative images of hematoxylin and eosin staining of the ileum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 0, 24, 48, 72, and 96 hours and pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Scale bar = 100 μm.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Inhibition, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Inhibition of the SHH pathway impairs intestinal epithelium regeneration in colon. Representative images of hematoxylin and eosin staining of the colon of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 0, 24, 48, 72, and 96 hours and pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Scale bar = 100 μm.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Inhibition, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Inhibition of the SHH pathway reduces postradiation MSI1 expression in vivo. Representative images of immunofluorescence staining for Bmi1-Cre ER -positive cells marker (YFP), proliferation marker (EdU), MSI1, and nuclei marker (DAPI) in the duodenum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 0, 24, 48, 72, and 96 hours and pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Scale bar = 100 μm.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Inhibition, Expressing, In Vivo, Immunofluorescence, Staining, Marker

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

doi: 10.1016/j.jcmgh.2023.07.004

Figure Lengend Snippet: Inhibition of the SHH pathway reduces postradiation SOX9 expression in vivo. Representative images of immunofluorescence staining for Bmi1-Cre ER -positive cells marker (YFP), WNT signaling marker (SOX9), MSI1, and nuclei marker (DAPI) in the duodenum of Bmi1-Cre ER mice exposed to 0 or 12 Gy TBI at 0, 24, 48, 72, and 96 hours and pretreated with vehicle (HBC) or SHH signaling inhibitor, CPN. Scale bar = 100 μm.

Article Snippet: To establish the biologically active dose of HH signaling pathway inhibitor, CPN (STEMCELL Technologies, Vancouver, Canada; #72074) cells were incubated with ADF medium supplemented with a biologically active dose of rSHH-N (0.25 μg/ml), and ADF medium containing either 0.2% ethanol or 5, 10, 20, or 50 μM CPN, respectively.

Techniques: Inhibition, Expressing, In Vivo, Immunofluorescence, Staining, Marker