Journal: PLoS ONE

Article Title: An Aquaporin 3-Notch1 Axis in Keratinocyte Differentiation and Inflammation

doi: 10.1371/journal.pone.0080179

Figure Lengend Snippet: (A) HKCs reverse transfected with siRNAs against CSL or scrambled control were analyzed 1 week later (in differentiating condition) by qRT-PCR and immunoblot of the indicated genes and proteins. mRNA levels were normalized to reference ribosomal gene RPLP0, and presented as mean fold-change over control ± S.E.M. n=3. ** p< 0.01, *** p < 0.001, **** p<0.0001, Hes1, Keratin 10 and Involucrin were used as indicators of endogenous Notch activity, γ-tubulin served as loading control. (B) Differentiating HKCs were treated with DAPT (10μM) or DMSO control for 72 hours followed by qRT-PCR and immunoblot analysis of the indicated genes and proteins. The analysis was done as in (A). **** p<0.0001, n=3. (C) Proliferating HKCs were infected with retroviruses expressing activated Notch1 or GFP followed, 72 hours later, by qRT-PCR and immunoblot analysis of AQP3. Hey1 was used as an indicator of endogenous Notch activity. The analysis was done as in (A). * p<0.05, *** p<0.001, n=3. (D) Chromatin immuno-precipitation (ChIP) analysis of CSL binding to the regulatory region of the AQP3 gene. Left panel: schematic representation of the AQP3 gene locus based on ChIP-seq information on different chromatin states as provided by the ENCODE project. The location of CSL binding site in the AQP3 gene was analyzed using Mat-inspector software. Right panel: HKCs were processed for ChIP analysis using CSL antibody or non immune IgG. Results are expressed as mean fold of enrichment ± S.E.M. n=2, for each indicated binding site, after immunoprecipitation with CSL antibody or non immune IgG, relative to enrichment for the negative control region.

Article Snippet: The processed chromatin was used for ChIP assays using the ChIP assay kit (Millipore) with CSL antibody (5313) from Cell Signaling.

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Activity Assay, Infection, Expressing, Chromatin Immunoprecipitation, Binding Assay, ChIP-sequencing, Software, Immunoprecipitation, Negative Control

Journal: PLoS ONE

Article Title: An Aquaporin 3-Notch1 Axis in Keratinocyte Differentiation and Inflammation

doi: 10.1371/journal.pone.0080179

Figure Lengend Snippet: (A) HKCs reverse transfected with siRNAs against CSL or scrambled control were analyzed 1 week later (in differentiating condition) by qRT-PCR and immunoblot of the indicated genes and proteins. mRNA levels were normalized to reference ribosomal gene RPLP0, and presented as mean fold-change over control ± S.E.M. n=3. ** p< 0.01, *** p < 0.001, **** p<0.0001, Hes1, Keratin 10 and Involucrin were used as indicators of endogenous Notch activity, γ-tubulin served as loading control. (B) Differentiating HKCs were treated with DAPT (10μM) or DMSO control for 72 hours followed by qRT-PCR and immunoblot analysis of the indicated genes and proteins. The analysis was done as in (A). **** p<0.0001, n=3. (C) Proliferating HKCs were infected with retroviruses expressing activated Notch1 or GFP followed, 72 hours later, by qRT-PCR and immunoblot analysis of AQP3. Hey1 was used as an indicator of endogenous Notch activity. The analysis was done as in (A). * p<0.05, *** p<0.001, n=3. (D) Chromatin immuno-precipitation (ChIP) analysis of CSL binding to the regulatory region of the AQP3 gene. Left panel: schematic representation of the AQP3 gene locus based on ChIP-seq information on different chromatin states as provided by the ENCODE project. The location of CSL binding site in the AQP3 gene was analyzed using Mat-inspector software. Right panel: HKCs were processed for ChIP analysis using CSL antibody or non immune IgG. Results are expressed as mean fold of enrichment ± S.E.M. n=2, for each indicated binding site, after immunoprecipitation with CSL antibody or non immune IgG, relative to enrichment for the negative control region.

Article Snippet: The processed chromatin was used for ChIP assays using the ChIP assay kit (Millipore) with CSL antibody (5313) from Cell Signaling.

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Activity Assay, Infection, Expressing, Chromatin Immunoprecipitation, Binding Assay, ChIP-sequencing, Software, Immunoprecipitation, Negative Control

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: RT-PCR primers.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques:

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: Effects of RPYD on α-SMA, TGF-β and Notch1 expression in lung tissue. (A) Immunohistochemical staining was used to assess the distribution of α-SMA, TGFβ1, Notch1 expression in lung tissue. The magnification was 200×. Scale bar: 50 μm. (B) Quantitative analysis of α-SMA positive area in airway smooth muscle. (C) Western blot analysis of TGFβ, Smad3, phospho-Smad3, Jagged1, Notch1, Hes1, α-SMA, E-cadherin protein levels with GAPDH as a standard control. (D) Results of band gray scale analysis of these proteins. (E) Quantitative RT-PCR was performed to determine the mRNA levels of TGFβ, Smad3, Notch1, Hes1, α-SMA, E-cadherin in lung tissue. Data are shown as mean ± SD ( n = 8). # P < 0.05 vs. control mice, * P < 0.05 vs. OVA mice.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Control, Quantitative RT-PCR

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: Effects of RPYD on Jagged1/Notch1 signaling in bronchial epithelial BEAS-2B cells induced by TGFβ1. BEAS-2B cells were treated as described in section “Materials and Methods.” (A) Western blot was used to detect the protein expression of Jagged1, Notch1, Hes1, α-SMA and E-cadherin. (B) Relative level of each protein. All data were shown as mean ± SD ( n = 3). # P < 0.05 vs. Control group, * P < 0.05 vs. TGFβ1 stimulation group.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: Effects of RPYD on α-SMA, TGF-β and Notch1 expression in lung tissue. (A) Immunohistochemical staining was used to assess the distribution of α-SMA, TGFβ1, Notch1 expression in lung tissue. The magnification was 200×. Scale bar: 50 μm. (B) Quantitative analysis of α-SMA positive area in airway smooth muscle. (C) Western blot analysis of TGFβ, Smad3, phospho-Smad3, Jagged1, Notch1, Hes1, α-SMA, E-cadherin protein levels with GAPDH as a standard control. (D) Results of band gray scale analysis of these proteins. (E) Quantitative RT-PCR was performed to determine the mRNA levels of TGFβ, Smad3, Notch1, Hes1, α-SMA, E-cadherin in lung tissue. Data are shown as mean ± SD ( n = 8). # P < 0.05 vs. control mice, * P < 0.05 vs. OVA mice.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Control, Quantitative RT-PCR

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: Effects of RPYD on Jagged1/Notch1 signaling in bronchial epithelial BEAS-2B cells induced by TGFβ1. BEAS-2B cells were treated as described in section “Materials and Methods.” (A) Western blot was used to detect the protein expression of Jagged1, Notch1, Hes1, α-SMA and E-cadherin. (B) Relative level of each protein. All data were shown as mean ± SD ( n = 3). # P < 0.05 vs. Control group, * P < 0.05 vs. TGFβ1 stimulation group.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: RT-PCR primers.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques:

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: Effects of RPYD on α-SMA, TGF-β and Notch1 expression in lung tissue. (A) Immunohistochemical staining was used to assess the distribution of α-SMA, TGFβ1, Notch1 expression in lung tissue. The magnification was 200×. Scale bar: 50 μm. (B) Quantitative analysis of α-SMA positive area in airway smooth muscle. (C) Western blot analysis of TGFβ, Smad3, phospho-Smad3, Jagged1, Notch1, Hes1, α-SMA, E-cadherin protein levels with GAPDH as a standard control. (D) Results of band gray scale analysis of these proteins. (E) Quantitative RT-PCR was performed to determine the mRNA levels of TGFβ, Smad3, Notch1, Hes1, α-SMA, E-cadherin in lung tissue. Data are shown as mean ± SD ( n = 8). # P < 0.05 vs. control mice, * P < 0.05 vs. OVA mice.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Control, Quantitative RT-PCR

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: Effects of RPYD on TGFβ1/Smad3 signaling in bronchial epithelial BEAS-2B cells induced by TGFβ1. BEAS-2B cells were treated as described in section “Materials and Methods.” (A) Western blot was used to detect the protein expression of phosphorylated Smad3, Smad3, α-SMA and E-cadherin. (B) Relative level of each protein. All data were shown as mean ± SD ( n = 3). # P < 0.05 vs. Control group, * P < 0.05 vs. TGFβ1 stimulation group. (C) Western blot results of cytosol and nuclear expression of Smad3. (D) Relative level of cytosol and nuclear Smad3.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: NICD overexpression promotes the phosphorylation and nuclear translocation of Smad3. (A) Expression of NICD in transfected cells was verified by RT-PCR and western blotting after BEAS-2B cells were infected with lentivirus for 24 h. All data were shown as mean ± SD ( n = 3). # P < 0.05 vs. Control group. (B) Western blot was used to detect phospho-Smad3, Smad3, and NICD protein expression. The band intensity of the protein was expressed as a ratio to GAPDH. All data were shown as mean ± SD ( n = 3). # P < 0.05 vs. Control group, * P < 0.05 vs. TGFβ1 stimulation group. (C) Western blot was used to detect Smad3, CSL, and Hes1 protein expression. The band intensity was expressed as a ratio to GAPDH or PARP. All data were shown as mean ± SD ( n = 3). # P < 0.05 vs. TGFβ1 stimulation group.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Over Expression, Phospho-proteomics, Translocation Assay, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Control

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: Smad3 siRNA reduces the level of Smad3 phosphorylation and decreases the protein expression of Hes1. (A) The specific knockdown of Smad3 by siRNA was verified by RT-PCR and western blotting. All data were shown as mean ± SD ( n = 3). # P < 0.05 vs. Control group. (B) Western blot was used to detect phospho-Smad3, Smad3, Hes1, and CSL protein expression. The band intensity was expressed as a ratio to GAPDH or PARP. All data were shown as mean ± SD ( n = 3). # P < 0.05 vs. Control group, * P < 0.05 vs. TGFβ1 stimulation group.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Phospho-proteomics, Expressing, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Frontiers in Physiology

Article Title: Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice

doi: 10.3389/fphys.2020.559470

Figure Lengend Snippet: RT-PCR primers.

Article Snippet: The antibodies of phosphor-Smad3 (#9520), Smad3 (#9523), Jagged1 (#70109), Notch1 (#3608), NICD (#4147), Hes1 (#11988), CSL (#5313), α-SMA (#19245), E-cadherin (#3195), GAPDH (#5174), and PARP (#9532) were all purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: