Journal: Cell biology and toxicology

Article Title: ASPP2 deficiency attenuates lipid accumulation through the PPARγ pathway in alcoholic liver injury.

doi: 10.1007/s10565-024-09925-x

Figure Lengend Snippet: Fig. 2 ASPP2 deficiency inhibits the PPARγ and mTOR signaling path- ways. (A and B) Repre- sentative IHC analysis of PPARγ in liver sections, scar bar = 50 µm (A) and quantification analyses (B) of PPARγ positive area (%) in mouse liver tissue from wild-type mice and ASPP2- KD mice. The extended part of the black lines shows the enlarged image from the black box area. (C and D) western blot (C) and quantification analyses (D) of ASPP2, PPARγ, phospho-mTOR, phospho- S6, phospho-p70S6K, and β-actin in mouse liver tissue from wild-type mice and ASPP2-KD mice. (E and F) western blot (E) and quantification analyses (F) of ASPP2, PPARγ, phospho-mTOR, phospho- S6, phospho-p70S6K, and β-actin in primary hepatocytes (Control and ASPP2 siRNA) treated with ethanol for different times. (G and H) western blot (G) and quantification analyses (H) of ASPP2, PPARγ, phospho-mTOR, phospho-S6, phospho- p70S6K, and β-actin in primary hepatocytes (Ad- GFP and Ad-ASPP2) with the indicated treatment. The values represent the means ± SEMs (n = 6 in each group). nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Independent- samples T tests between two groups were used for statistical analysis, and one-way ANOVA followed by Bonferroni post hoc tests for multiple comparisons were used for statistical analyses

Article Snippet: Finally, the expression of ASPP2 or PPARγ was observed by microscopy (Leica Microsystems, Mannheim, Germany) after development using a diaminobenzidine kit (Boster, Wuhan, China).

Techniques: Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: ASPP2 is increased in the livers of ALI patients and mouse models induced by D-GalN/LPS. (A–C) Liver tissue obtained from normal controls, patients with CHB and HBV-ALI were analyzed the expression level of ASPP2. (A) Gene expressions of ASPP2 was measured by qRT-PCR. (B) Protein expression levels of ASPP2 was measured by western blot assays. (C) The expression of ASPP2 was measured by immunofluorescence staining. (D–F) Mice were intraperitoneally injected with D-GalN (700 mg/kg) and LPS (10 μg/kg) at 2-, 4- and 6- hours (12 mice/group). The mice in the control group (n = 8) were injected with PBS only. (D) Serum AST and ALT enzyme levels. (E) Gene expressions of ASPP2 was measured by qRT-PCR. (F) Protein expression levels of ASPP2 was measured by western blot assays. *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Then, incubated with ASPP2 mouse polyclonal antibodies (1:200, Abcam, Cambridge, MA) diluted in 3% BSA overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Injection

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: Knockdown of ASPP2 protects against D-GalN/LPS-induced acute liver injury. ASPP2 +/+ mice and ASPP2 +/- mice were intraperitoneally injected with D-GalN/LPS for 6 hours (12 mice/group). (A) Representative livers and H&E staining of livers from different groups. (B) Serum AST and ALT enzyme levels from different groups. (C) Measurement of cytokine levels in the serum of mice in different groups by the Luminex Milliplex ® MAP Kit (TNF-α, IL-6, IL-1β, IL-18, IL-12, IL-10, CXCL-10, IL-13, IL-22). (D) Analysis of the gene expression levels of cytokines (TNF-α, IL-6, IL-1β, IL-18, IL-10, IL-12, CXCL-10) in liver tissues from different groups by qRT-PCR. (E) TUNEL staining (red) liver tissue at 6 h after D-GalN/LPS administration. Original magnification×200. (F) Caspase 3 activity of livers was measured from different groups. (G) Protein expression levels of p-JNK, caspase 3 and cleaved caspase 3 was measured by western blot assays. *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Then, incubated with ASPP2 mouse polyclonal antibodies (1:200, Abcam, Cambridge, MA) diluted in 3% BSA overnight at 4°C.

Techniques: Injection, Staining, Luminex, Expressing, Quantitative RT-PCR, TUNEL Assay, Activity Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: Knockdown of ASPP2 reduced hepatocyte apoptosis through autophagy activation during ALI. (A, B) ASPP2 +/+ mice and ASPP2 +/- mice were intraperitoneally injected with D-GalN/LPS for 6 hours (12 mice/group). (A) Autophagosomes was observed by electron microscope in the livers from different groups. (B) Protein expression levels of LC3, p62, ATG-5, Atg-7 and Beclin-1 were measured by western blotting. (C–E) Mice were pretreated with Atg7 or negative control siRNA (3mg/kg, iv) for 48 hours prior to D-GalN/LPS exposure (n = 12). (C) Serum AST and ALT enzyme levels from different groups. (D) TUNEL staining (red) liver tissue from different groups. Original magnification×200. (E) Caspase 3 activity of livers was measured from different groups. *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Then, incubated with ASPP2 mouse polyclonal antibodies (1:200, Abcam, Cambridge, MA) diluted in 3% BSA overnight at 4°C.

Techniques: Activation Assay, Injection, Microscopy, Expressing, Western Blot, Negative Control, TUNEL Assay, Staining, Activity Assay

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: ASPP2-depleted BMMs protect hepatocytes from apoptosis by reducing TNF-α levels in vitro. (A, B) Primary hepatocytes were treated with conditioned medium (CM) of ASPP2 +/+ BMMs or ASPP2 +/- BMMs that with or without LPS (20ng/ml). (A, B) The cell viability and cell death rate of primary hepatocytes were measured by CCK-8 assays and LDH assays, respectively. (C) The cell apoptosis rate of primary hepatocytes was measured by flow cytometry. (D) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 6 hours, the level of TNF-α was analyzed by qRT-PCR. (E) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) as the indicated time points, the level of TNF-α was analyzed by qRT-PCR. (F) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 6 hours, the level of TNF-α in medium was analyzed by ELISA. *p<0.1, ***p<0.001, ****p<0.0001.

Article Snippet: Then, incubated with ASPP2 mouse polyclonal antibodies (1:200, Abcam, Cambridge, MA) diluted in 3% BSA overnight at 4°C.

Techniques: In Vitro, CCK-8 Assay, Flow Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: ASPP2-depleted BMMs decreased TNF-α by up-regulation of IL-6. (A) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 6 hours, the level of IL-6 was analyzed by qRT-PCR. E. ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) as the indicated time points, the level of IL-6 was analyzed by qRT-PCR. E. ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 6 hours, the level of IL-6 in medium was analyzed by ELISA. (D) IL-6 siRNA or negative control siRNA was transected into ASPP2 +/+ BMMs and ASPP2 +/- BMMs that with or without LPS (20ng/ml) stimulation. The level of TNF-α in BMMs were analyzed by qRT-PCR. (E, F) primary hepatocytes were treated with conditioned medium (CM) collected from (D) . The cell viability and cell death rate of primary hepatocytes were measured by CCK-8 assays and LDH assays, respectively. (G) Primary hepatocytes were treated with CM the same as (D) . The cell apoptosis rate of primary hepatocytes was measured by flow cytometry. *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Then, incubated with ASPP2 mouse polyclonal antibodies (1:200, Abcam, Cambridge, MA) diluted in 3% BSA overnight at 4°C.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Negative Control, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: ASPP2 +/− BMMs promote IL-6 expression through autophagy induction. (A, B) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 12 hours. (A) The gene expression levels of autophagy related genes (LC3, ATG-7, ATG-5, Beclin-1) were analyzed by qRT-PCR. (B) The protein expression levels of autophagy related genes (LC3, ATG-7, ATG-5, Beclin-1) were analyzed by western blotting. (C) DMSO and 3-MA were pretreated with cells 2 hours before LPS stimulation. The gene expression levels of cytokines (IL-6, TNF-α) in cells were analyzed by qRT-PCR. (D, E) Primary hepatocytes were incubated with conditioned medium (CM) of BMMs treated as (C) The cell viability and cell death rate of primary hepatocytes were measured by CCK-8 assays and LDH assays, respectively. (F) The cell apoptosis rate of primary hepatocytes was measured by flow cytometry. *p<0.1, **p<0.01, ****p<0.0001.

Article Snippet: Then, incubated with ASPP2 mouse polyclonal antibodies (1:200, Abcam, Cambridge, MA) diluted in 3% BSA overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Incubation, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: Upregulation of ASPP2 sensitizes hepatocytes to TNF-α-induced apoptosis. (A-C) Primary hepatocytes were transfected with ASPP2 over-expression (OE) plasmid or ASPP2 siRNA, and treated with TNF-α for 12 hours. (A, B) The cell viability and cell death rate of primary hepatocytes were measured by CCK-8 assays and LDH assays, respectively. (C) The cell apoptosis rate of primary hepatocytes was measured by flow cytometry. (D) The graphic abstract of this study. In ALI mice induced by D-GalN/LPS, ASPP2 was increased in liver Kupffer cells, which suppressed the level of IL-6 and increased the level of TNF-α through inhibition autophagy to promote hepatocytes apoptosis. In addition, upregulation of ASPP2 in hepatocytes promoted cell apoptosis induced by TNF-α. *p<0.1, ***p<0.001, ****p<0.0001.

Article Snippet: Then, incubated with ASPP2 mouse polyclonal antibodies (1:200, Abcam, Cambridge, MA) diluted in 3% BSA overnight at 4°C.

Techniques: Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Inhibition

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: ASPP2 is increased in the livers of ALI patients and mouse models induced by D-GalN/LPS. (A–C) Liver tissue obtained from normal controls, patients with CHB and HBV-ALI were analyzed the expression level of ASPP2. (A) Gene expressions of ASPP2 was measured by qRT-PCR. (B) Protein expression levels of ASPP2 was measured by western blot assays. (C) The expression of ASPP2 was measured by immunofluorescence staining. (D–F) Mice were intraperitoneally injected with D-GalN (700 mg/kg) and LPS (10 μg/kg) at 2-, 4- and 6- hours (12 mice/group). The mice in the control group (n = 8) were injected with PBS only. (D) Serum AST and ALT enzyme levels. (E) Gene expressions of ASPP2 was measured by qRT-PCR. (F) Protein expression levels of ASPP2 was measured by western blot assays. *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies against LC3B, Atg7, Atg5, Beclin-1, p62, β-actin (Cell Signaling Technology Inc., Santa Cruz, CA, USA), ASPP2 (Sigma, St. Louis, MO, USA) were used for western blot analysis.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Injection

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: Knockdown of ASPP2 protects against D-GalN/LPS-induced acute liver injury. ASPP2 +/+ mice and ASPP2 +/- mice were intraperitoneally injected with D-GalN/LPS for 6 hours (12 mice/group). (A) Representative livers and H&E staining of livers from different groups. (B) Serum AST and ALT enzyme levels from different groups. (C) Measurement of cytokine levels in the serum of mice in different groups by the Luminex Milliplex ® MAP Kit (TNF-α, IL-6, IL-1β, IL-18, IL-12, IL-10, CXCL-10, IL-13, IL-22). (D) Analysis of the gene expression levels of cytokines (TNF-α, IL-6, IL-1β, IL-18, IL-10, IL-12, CXCL-10) in liver tissues from different groups by qRT-PCR. (E) TUNEL staining (red) liver tissue at 6 h after D-GalN/LPS administration. Original magnification×200. (F) Caspase 3 activity of livers was measured from different groups. (G) Protein expression levels of p-JNK, caspase 3 and cleaved caspase 3 was measured by western blot assays. *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies against LC3B, Atg7, Atg5, Beclin-1, p62, β-actin (Cell Signaling Technology Inc., Santa Cruz, CA, USA), ASPP2 (Sigma, St. Louis, MO, USA) were used for western blot analysis.

Techniques: Injection, Staining, Luminex, Expressing, Quantitative RT-PCR, TUNEL Assay, Activity Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: Knockdown of ASPP2 reduced hepatocyte apoptosis through autophagy activation during ALI. (A, B) ASPP2 +/+ mice and ASPP2 +/- mice were intraperitoneally injected with D-GalN/LPS for 6 hours (12 mice/group). (A) Autophagosomes was observed by electron microscope in the livers from different groups. (B) Protein expression levels of LC3, p62, ATG-5, Atg-7 and Beclin-1 were measured by western blotting. (C–E) Mice were pretreated with Atg7 or negative control siRNA (3mg/kg, iv) for 48 hours prior to D-GalN/LPS exposure (n = 12). (C) Serum AST and ALT enzyme levels from different groups. (D) TUNEL staining (red) liver tissue from different groups. Original magnification×200. (E) Caspase 3 activity of livers was measured from different groups. *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies against LC3B, Atg7, Atg5, Beclin-1, p62, β-actin (Cell Signaling Technology Inc., Santa Cruz, CA, USA), ASPP2 (Sigma, St. Louis, MO, USA) were used for western blot analysis.

Techniques: Activation Assay, Injection, Microscopy, Expressing, Western Blot, Negative Control, TUNEL Assay, Staining, Activity Assay

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: ASPP2-depleted BMMs protect hepatocytes from apoptosis by reducing TNF-α levels in vitro. (A, B) Primary hepatocytes were treated with conditioned medium (CM) of ASPP2 +/+ BMMs or ASPP2 +/- BMMs that with or without LPS (20ng/ml). (A, B) The cell viability and cell death rate of primary hepatocytes were measured by CCK-8 assays and LDH assays, respectively. (C) The cell apoptosis rate of primary hepatocytes was measured by flow cytometry. (D) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 6 hours, the level of TNF-α was analyzed by qRT-PCR. (E) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) as the indicated time points, the level of TNF-α was analyzed by qRT-PCR. (F) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 6 hours, the level of TNF-α in medium was analyzed by ELISA. *p<0.1, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies against LC3B, Atg7, Atg5, Beclin-1, p62, β-actin (Cell Signaling Technology Inc., Santa Cruz, CA, USA), ASPP2 (Sigma, St. Louis, MO, USA) were used for western blot analysis.

Techniques: In Vitro, CCK-8 Assay, Flow Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: ASPP2-depleted BMMs decreased TNF-α by up-regulation of IL-6. (A) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 6 hours, the level of IL-6 was analyzed by qRT-PCR. E. ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) as the indicated time points, the level of IL-6 was analyzed by qRT-PCR. E. ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 6 hours, the level of IL-6 in medium was analyzed by ELISA. (D) IL-6 siRNA or negative control siRNA was transected into ASPP2 +/+ BMMs and ASPP2 +/- BMMs that with or without LPS (20ng/ml) stimulation. The level of TNF-α in BMMs were analyzed by qRT-PCR. (E, F) primary hepatocytes were treated with conditioned medium (CM) collected from (D) . The cell viability and cell death rate of primary hepatocytes were measured by CCK-8 assays and LDH assays, respectively. (G) Primary hepatocytes were treated with CM the same as (D) . The cell apoptosis rate of primary hepatocytes was measured by flow cytometry. *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies against LC3B, Atg7, Atg5, Beclin-1, p62, β-actin (Cell Signaling Technology Inc., Santa Cruz, CA, USA), ASPP2 (Sigma, St. Louis, MO, USA) were used for western blot analysis.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Negative Control, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: ASPP2 +/− BMMs promote IL-6 expression through autophagy induction. (A, B) ASPP2 +/+ BMMs or ASPP2 +/- BMMs were treated with LPS (20ng/ml) for 12 hours. (A) The gene expression levels of autophagy related genes (LC3, ATG-7, ATG-5, Beclin-1) were analyzed by qRT-PCR. (B) The protein expression levels of autophagy related genes (LC3, ATG-7, ATG-5, Beclin-1) were analyzed by western blotting. (C) DMSO and 3-MA were pretreated with cells 2 hours before LPS stimulation. The gene expression levels of cytokines (IL-6, TNF-α) in cells were analyzed by qRT-PCR. (D, E) Primary hepatocytes were incubated with conditioned medium (CM) of BMMs treated as (C) The cell viability and cell death rate of primary hepatocytes were measured by CCK-8 assays and LDH assays, respectively. (F) The cell apoptosis rate of primary hepatocytes was measured by flow cytometry. *p<0.1, **p<0.01, ****p<0.0001.

Article Snippet: Antibodies against LC3B, Atg7, Atg5, Beclin-1, p62, β-actin (Cell Signaling Technology Inc., Santa Cruz, CA, USA), ASPP2 (Sigma, St. Louis, MO, USA) were used for western blot analysis.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Incubation, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism

doi: 10.3389/fimmu.2024.1381735

Figure Lengend Snippet: Upregulation of ASPP2 sensitizes hepatocytes to TNF-α-induced apoptosis. (A-C) Primary hepatocytes were transfected with ASPP2 over-expression (OE) plasmid or ASPP2 siRNA, and treated with TNF-α for 12 hours. (A, B) The cell viability and cell death rate of primary hepatocytes were measured by CCK-8 assays and LDH assays, respectively. (C) The cell apoptosis rate of primary hepatocytes was measured by flow cytometry. (D) The graphic abstract of this study. In ALI mice induced by D-GalN/LPS, ASPP2 was increased in liver Kupffer cells, which suppressed the level of IL-6 and increased the level of TNF-α through inhibition autophagy to promote hepatocytes apoptosis. In addition, upregulation of ASPP2 in hepatocytes promoted cell apoptosis induced by TNF-α. *p<0.1, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies against LC3B, Atg7, Atg5, Beclin-1, p62, β-actin (Cell Signaling Technology Inc., Santa Cruz, CA, USA), ASPP2 (Sigma, St. Louis, MO, USA) were used for western blot analysis.

Techniques: Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Inhibition

Journal: Cell Insight

Article Title: Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

doi: 10.1016/j.cellin.2024.100161

Figure Lengend Snippet: Characterization of cortactin gene knockout in intestinal Caco-2 cells by CRISPR-Cas9. ( A ) Confirmation of cortactin gene knockout in Caco-2Δ cttn cells by fluorescence microscopy using cortactin-specific antibodies (green) and detecting the functional HDR plasmid, expressing RFP (red). Immunostaining of ZO-1 and occludin served as controls. ( B ) Western blot showing a ∼85 kDa protein corresponding to cortactin in Caco-2 wt cells, but not in Caco-2Δ cttn mutant clones 2 and 6. Western blotting with antibodies specific for ZO-1, E-cadherin, Claudin-5, ASPP2 and β-actin served as control. ( C ) Phase-contrast microscopy of representative Caco-2 wt and Caco-2Δ cttn cell lines showing cellular morphologies within respective monolayer. Quantification of cell ( D ) and nuclear ( E ) areas based on F-actin and nuclear staining, respectively, displaying arithmetic means ± SD (standard deviation) as well as individual values (dots). The size differences between Caco-2 wt and Caco-2Δ cttn cells were confirmed to be statistically significant with p ​< ​0.001 (∗∗∗).

Article Snippet: Membranes were blocked with either 5% non-fat milk or 3% BSA and probed with the primary antibodies as follows: mouse α-cortactin (#05–180, Merck-Millipore), rabbit α-Par1b (#HPA074905, Sigma Aldrich), rabbit α-ZO-1 (#61–73000, Invitrogen), rabbit α-E-cadherin (#sc-7870, Santa Cruz), rabbit α-Claudin-5 (#ab15106, Abcam), mouse α-β-actin (#A5441, Sigma Aldrich), mouse α-GFP (#632381, Clontech), rabbit α-omni-probe (α-T7) (#sc-499, Santa Cruz), rabbit α-CagA (#HPP-5003-9, Austral Biologicals), mouse α-PY99 (#sc-7020, Santa Cruz) and α-ASPP2 (#200-401-A19, Rockland).

Techniques: Gene Knockout, CRISPR, Fluorescence, Microscopy, Functional Assay, Plasmid Preparation, Expressing, Immunostaining, Western Blot, Mutagenesis, Clone Assay, Control, Staining, Standard Deviation

Journal: Cell Insight

Article Title: Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

doi: 10.1016/j.cellin.2024.100161

Figure Lengend Snippet: H. pylori CagA promotes complex formation of cortactin with ZO-1 . ( A ) Fluorescence microscopy of Caco-2 wt cell monolayer cross-sections (X/Z dimension) without or after infection with H. pylori wt or Δ virB10 mutant. The white dashed lines indicate apical and basal surfaces of the monolayers. Yellow arrows indicate abnormal localization of ZO-1. ( B ) Western blot analysis of protein complex formation in Caco-2 wt cells after H. pylori infection (MOI 100) for 6 h by IP using α-ZO-1 antibodies. Proteins co-immunoprecipitated with ZO-1 were probed using antibodies against cortactin, CagA, β-actin and ASPP2. ( C ) The band intensities of cortactin, β-actin and CagA immunoprecipitated in a complex with ZO-1 were quantified and expressed as relative, protein complex-bound cortactin, β-actin or CagA. Mean intensities ​± ​SD are presented; p ​< ​0.001 (∗∗∗).

Article Snippet: Membranes were blocked with either 5% non-fat milk or 3% BSA and probed with the primary antibodies as follows: mouse α-cortactin (#05–180, Merck-Millipore), rabbit α-Par1b (#HPA074905, Sigma Aldrich), rabbit α-ZO-1 (#61–73000, Invitrogen), rabbit α-E-cadherin (#sc-7870, Santa Cruz), rabbit α-Claudin-5 (#ab15106, Abcam), mouse α-β-actin (#A5441, Sigma Aldrich), mouse α-GFP (#632381, Clontech), rabbit α-omni-probe (α-T7) (#sc-499, Santa Cruz), rabbit α-CagA (#HPP-5003-9, Austral Biologicals), mouse α-PY99 (#sc-7020, Santa Cruz) and α-ASPP2 (#200-401-A19, Rockland).

Techniques: Fluorescence, Microscopy, Infection, Mutagenesis, Western Blot, Immunoprecipitation

Journal: Cell Insight

Article Title: Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

doi: 10.1016/j.cellin.2024.100161

Figure Lengend Snippet: H. pylori deregulates the host proteins cortactin, Par1b and ZO-1 in human mucosoids via CagA. ( A ) Fluorescence microscopy of human mucosoids after 6 h infection with either H. pylori wt or Δ cagA mutant. Polarized mucosoids were cultured in a transwell system followed by fixation in PFA and staining with phalloidin to visualize F-actin structures. ( B ) Immunofluorescence microscopy of mucosoid cross-sections (X/Z dimension) after 6 h infection with either H. pylori wt or Δ cagA mutant. ( C ) Z-profiles of Par1b, cortactin, F-actin and DAPI fluorescence from the mucosoid cross-section of representative cells (indicated by yellow arrows in panel B ) show the basal and apical distributions of proteins; a.u. – arbitrary units ( D - E ) Human mucosoids were infected with H. pylori wt or either Δ cagA or Δ virB7 mutants. The samples were immunoprecipitated with α-ZO-1 ( D ) or α-Par1b ( E ) antibodies, and analyzed by Western blotting using antibodies against ZO-1, Par1b, cortactin, CagA and ASPP2.

Article Snippet: Membranes were blocked with either 5% non-fat milk or 3% BSA and probed with the primary antibodies as follows: mouse α-cortactin (#05–180, Merck-Millipore), rabbit α-Par1b (#HPA074905, Sigma Aldrich), rabbit α-ZO-1 (#61–73000, Invitrogen), rabbit α-E-cadherin (#sc-7870, Santa Cruz), rabbit α-Claudin-5 (#ab15106, Abcam), mouse α-β-actin (#A5441, Sigma Aldrich), mouse α-GFP (#632381, Clontech), rabbit α-omni-probe (α-T7) (#sc-499, Santa Cruz), rabbit α-CagA (#HPP-5003-9, Austral Biologicals), mouse α-PY99 (#sc-7020, Santa Cruz) and α-ASPP2 (#200-401-A19, Rockland).

Techniques: Fluorescence, Microscopy, Infection, Mutagenesis, Cell Culture, Staining, Immunofluorescence, Immunoprecipitation, Western Blot

Journal: Cell Insight

Article Title: Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

doi: 10.1016/j.cellin.2024.100161

Figure Lengend Snippet: Models highlighting the importance of cortactin in regulating cell polarity through tight junctions (TJ). ( A ) A simplified overview shows that wild-type epithelial monolayers exhibit normal cell polarity provided directly by the Par polarity proteins and indirectly by cortactin. Par1b mainly locates to the basal membrane, as expected. In addition, cortactin seems to regulate monolayer permeability, presumably via the apical junctional complex, in particular by binding to ZO-1. This binding requires serine-phosphorylation of cortactin. ( B ) In cortactin-deficient cells, Par1b mainly locates to the apical membranes leading to disturbed cell polarity. We therefore propose that cortactin exhibits some suppressive activity on Par1b in the TJs. Thus, elevated TEER values were measured that may arise from the absence of cortactin. In addition, the expression of cortactin seems important for proper microvilli formation. ( C ) In H. pylori -infected wild-type monolayers, injected CagA induces Par1b inactivation and serine-phosphorylation of cortactin, associated with strongly enhanced complex formation with ZO-1 in the TJs, which leads to loss of cell polarity. ( D ) During infection with H. pylori, CagA is injected into epithelial cells, which targets Par1b and cell polarity by two different pathways, here named complex-1 and complex-2. In complex-1, CagA binds directly to Par1b via the CRPIA-motif, which triggers loss of cell polarity and may promote cell extrusion. This complex also contains ZO-1 and cortactin. In complex-2, CagA targets ASPP2 at the ABD (ASPP2 Binding Domain) to inhibit the aPKC-containing apical regulatory complex. In this way, aPKC cannot phosphorylate Par3 ( Buti et al., 2020 ) and CagA-bound Par1b ( Saadat et al., 2007 ) any longer, which abrogates their mutual antagonistic activities. We propose that both signaling complexes together result in full disruption of cell polarity and induction of cytoskeletal rearrangements.

Article Snippet: Membranes were blocked with either 5% non-fat milk or 3% BSA and probed with the primary antibodies as follows: mouse α-cortactin (#05–180, Merck-Millipore), rabbit α-Par1b (#HPA074905, Sigma Aldrich), rabbit α-ZO-1 (#61–73000, Invitrogen), rabbit α-E-cadherin (#sc-7870, Santa Cruz), rabbit α-Claudin-5 (#ab15106, Abcam), mouse α-β-actin (#A5441, Sigma Aldrich), mouse α-GFP (#632381, Clontech), rabbit α-omni-probe (α-T7) (#sc-499, Santa Cruz), rabbit α-CagA (#HPP-5003-9, Austral Biologicals), mouse α-PY99 (#sc-7020, Santa Cruz) and α-ASPP2 (#200-401-A19, Rockland).

Techniques: Membrane, Permeability, Binding Assay, Phospho-proteomics, Activity Assay, Expressing, Infection, Injection, Disruption