immunofluorescence triple staining  (SLIT2 LTD)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: Restoration of hepatic MBL expression eliminates MBL absence–mediated reduction of senescent HSC frequency. The MBL -/- and WT mice (n = 5 per group) received a pAAV-control or pAAV-MBL vector injection 3 weeks before liver fibrosis establishment. ( A ) Representative immunofluorescence staining of α-SMA (green) and p21 (red), p16 (red), p-H2.X (red), or Ki67 (red) in liver tissues. ( B ) The p53 and p21 protein levels in liver tissues were assessed by Western blot analysis. ( C ) The mRNA levels of senescence-related genes in liver tissues were assessed by quantitative reverse-transcription PCR analysis. Scale bars : 100 μm. Data are presented as means ± SEM. ∗∗ P < .01, 1-way analysis of variance followed by Tukey post hoc tests for multiple group comparisons. CCL, chemokine (C-C motif) ligand; CXCL, chemokine (C-X-C motif) ligand; DAPI, 4′,6-diamidino-2-phenylindole; HPF, high-power field; IL, interleukin; MMP, matrix metalloproteinase.

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: Expressing, Control, Plasmid Preparation, Injection, Immunofluorescence, Staining, Western Blot, Reverse Transcription

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: Senolytic treatment eliminates senescent cells in mice fibrotic livers. The MBL -/- and WT mice (n = 6 per group) were garaged with D+Q every 3 weeks for 3 times during the fibrosis establishment. ( A ) Representative immunofluorescence staining of α-SMA (green) and p16 (red), p-H2.X (red), or Ki67 (red) in liver tissues. ( B ) Representative immunofluorescence staining of α-SMA (red) and Bax (green) or Bcl2 (green) in liver tissues. ( C ) The p53 and p21 protein levels in liver tissues were assessed by Western blot analysis. ( D ) The mRNA levels of senescence-related genes in liver tissues were assessed by quantitative reverse-transcription PCR analysis. Scale bars : 100 μm. Data are presented as means ± SEM. ∗ P < .05, ∗∗ P < .01, 1-way analysis of variance followed by Tukey post hoc tests for multiple group comparisons. CCL, chemokine (C-C motif) ligand; CXCL, chemokine (C-X-C motif) ligand; DAPI, 4′,6-diamidino-2-phenylindole; HPF, high-power field; IL, interleukin; MMP, matrix metalloproteinase.

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: Immunofluorescence, Staining, Western Blot, Reverse Transcription

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: MBL co-localizes with senescent HSCs in vivo. ( A ) Representative photomicrographs of colocalization of MBL (red) and α-SMA (green) in liver tissues of WT mice by immunofluorescence analysis. Scale bars : 50 μm. ( B ) Representative photomicrographs of colocalization of α-SMA (red), MBL (green), and p53 (purple) expression in liver tissues of cirrhotic patients by immunofluorescence analysis. Scale bars : 25 μm. Data are presented as means ± SEM. ∗∗ P < .01, Student t test. DAPI, 4′,6-diamidino-2-phenylindole; HPF, high-power field.

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: In Vivo, Immunofluorescence, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: MBL prompts senescence of HSCs in vitro. LX-2 cells were treated with MBL (10 μg/mL) and/or H 2 O 2 (300 μmol/L) for 48 hours. ( A ) Representative images of the SA–β-gal staining of LX-2 cells treated with or without MBL and/or H 2 O 2 . ( B ) The distribution of cell cycle on the treated LX-2 cells with flow cytometry analysis. ( C ) The p53 and p21 protein levels were assessed by Western blot analysis. ( D ) The mRNA levels of senescence-related genes were assessed by quantitative reverse-transcription (qRT)-PCR analysis. ( E ) The mRNA levels of fibrosis-associated genes were assessed by qRT-PCR analysis. ( F ) The apoptosis on the treated LX-2 cells with flow cytometry analysis. Scale bars : 100 μm. Data are presented as means ± SEM. ∗ P < .05, ∗∗ P < .01, 1-way analysis of variance followed by Tukey post hoc tests for multiple group comparisons. The data shown represent 3 independent experiments. CON, control; CXCL, chemokine (C-X-C motif) ligand; HPF, high-power field; IL, interleukin; MMP, matrix metalloproteinase; TIMP1, tissue inhibitors of metalloproteinase 1.

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: In Vitro, Staining, Flow Cytometry, Western Blot, Reverse Transcription, Quantitative RT-PCR, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: Rapamycin treatment abrogates MBL-mediated amelioration of liver fibrosis. WT and MBL -/- mice (n = 5 per group) were treated with pAAV-control or pAAV-MBL vectors before the establishment of liver fibrosis. ( A ) The protein levels of p110, mTOR, p-mTOR, AKT, and p-AKT in liver tissues were assessed by Western blot analysis. The MBL -/- and WT mice (n = 5 per group) were injected intraperitoneally with rapamycin (1 mg/kg body weight/d) during the fibrosis establishment. ( B ) The protein levels of mTOR, p-mTOR, p53, and p21 in mouse liver were assessed by Western blot analysis. ( C ) The mRNA levels of p53, p21, α-SMA, and collagen 1 were assessed by quantitative reverse-transcription PCR. ( D ) Representative immunofluorescence staining of α-SMA (green) and p21 (red) in liver sections. ( E ) Representative photomicrographs of H&E, Sirius Red, and Masson’s trichrome staining in the liver tissues. Scale bars : 100 μm. Data are presented as means ± SEM. ∗∗ P < .01, 1-way analysis of variance followed by Tukey post hoc tests for multiple group comparisons. DAPI, 4′,6-diamidino-2-phenylindole; HPF, high-power field.

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: Control, Western Blot, Injection, Reverse Transcription, Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: MBL prompts HSC senescence via the p53/p21 pathway. LX-2 cells were treated with PFT-α (10 μmol/L) 2 hours before the incubation of MBL and H 2 O 2 . ( A ) Representative photomicrographs of the SA–β-gal staining of LX-2 cells treated with PFT before the incubation of MBL. ( B ) The distribution of cell cycle on the treated LX-2 cells with flow cytometry analysis. ( C ) The p53 and p21 protein levels were assessed by Western blot analysis. ( D ) The mRNA levels of senescence-related genes were assessed by quantitative reverse-transcription (qRT)-PCR analysis. ( E ) The mRNA levels of fibrosis-associated genes were assessed by qRT-PCR analysis. Scale bars : 100 μm. Data are presented as means ± SEM. ∗∗ P < .01, 1-way analysis of variance followed by Tukey post hoc tests for multiple group comparisons. The data shown represent 3 independent experiments. CXCL, chemokine (C-X-C motif) ligand; HPF, high-power field; IL, interleukin; MMP, matrix metalloproteinase; TIMP1, tissue inhibitors of metalloproteinase 1.

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: Incubation, Staining, Flow Cytometry, Western Blot, Reverse Transcription, Quantitative RT-PCR

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: The mTOR/p53/p21 pathway is involved in the MBL-mediated promotion of HSC senescence. LX-2 cells were treated with rapamycin (100 nmol/L) 2 hours before the incubation of MBL and H 2 O 2 . ( A ) Representative photomicrographs of the SA–β-gal staining of LX-2 cells treated with rapamycin before the incubation of MBL. ( B ) The distribution of cell cycle on the treated LX-2 cells with flow cytometry analysis. ( C ) The p53, p21, mTOR, and p-mTOR protein levels were assessed by Western blot analysis. ( D ) The mRNA levels of senescence-related genes were assessed by quantitative reverse-transcription (qRT)-PCR analysis. ( E ) The mRNA levels of fibrosis-associated genes were assessed by qRT-PCR analysis. Scale bars : 100 μm. Data are presented as means ± SEM. ∗∗ P < .01, 1-way analysis of variance followed by Tukey post hoc tests for multiple group comparisons. The data shown represent 3 independent experiments. CCL, chemokine (C-C motif) ligand; CXCL, chemokine (C-X-C motif) ligand; HPF, high-power field; IL, interleukin; MMP, matrix metalloproteinase.

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: Incubation, Staining, Flow Cytometry, Western Blot, Reverse Transcription, Quantitative RT-PCR

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: MBL binds to CRT on the cell surface of HSCs. ( A ) LX-2 cells were incubated with MBL protein and the membrane fractions were extracted for subsequent immunoprecipitation. The association of MBL with CRT was determined by immunoblotting of immunoprecipitates with anti-CRT. ( B ) HSCs were extracted from the mouse liver and cultured for 14 days, followed by incubation with primary hepatocyte supernatants. The protein levels of α-SMA of HSCs on day 5 and day 14. ( C ) Representative immunofluorescence staining of α-SMA (green) in HSCs on day 14. ( D ) Representative immunofluorescence staining of α-SMA (green) and MBL (red) in HSCs stimulated with primary hepatocytes supernatants. ( E ) The protein levels of mTOR, p-mTOR, p53, and p21 in HSCs stimulated with primary hepatocyte supernatants. Scale bars : 25 μm. The data shown represent 3 independent experiments. ATPase, adenosine triphosphatase; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: Incubation, Membrane, Immunoprecipitation, Western Blot, Cell Culture, Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: MBL–CRT interaction up-regulates the association of CRT and LRP1 and downstream senescent-related signaling pathways in HSC. LX-2 cells were treated with CRT antibody or IgG 1 hour before the incubation of MBL and H 2 O 2 . ( A ) Representative photomicrographs of the SA–β-gal staining of LX-2 cells. ( B ) The distribution of cell cycle on the treated LX-2 cells with flow cytometry analysis. ( C ) The mRNA levels of senescence-related genes were assessed by quantitative reverse-transcription (qRT)-PCR analysis. ( D ) The association of CRT and LRP1 was determined by immunoblotting of immunoprecipitates in membrane fractions of LX-2 cells. ( E ) The association of LRP1, Rab8, and p110 was determined by immunoblotting of immunoprecipitates in cytoplasm fractions of LX-2 cells. ( F ) The p53, p21, mTOR, p-mTOR, AKT, p-AKT, and p110 protein levels were assessed by Western blot analysis. ( G ) The mRNA levels of fibrosis-associated genes were assessed by qRT-PCR analysis. Scale bars : 100 μm. Data are presented as means ± SEM. ∗∗ P < .01, Student t test or 1-way analysis of variance followed by Tukey post hoc tests for multiple group comparisons. The data shown represent 3 independent experiments. ATPase, adenosine triphosphatase; CCL, chemokine (C-C motif) ligand; CXCL, chemokine (C-X-C motif) ligand; HPF, high-power field; IL, interleukin; MMP, matrix metalloproteinase.

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: Protein-Protein interactions, Incubation, Staining, Flow Cytometry, Reverse Transcription, Quantitative RT-PCR, Western Blot, Membrane

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: Primers for Mice and Human Beings

Article Snippet: The triple immunofluorescent staining of p53, MBL, and α-SMA on liver sections was conducted by Servicebio Company (Wuhan, China).

Techniques: