mouse anti-hrv-a16 antibody  (QED Bioscience)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells

doi: 10.3389/fcimb.2020.00278

Figure Lengend Snippet: Primer sequences.

Article Snippet: PBS/1% (w/v) BSA/0.3% (w/v) Triton-X-100 (PBT) was used to block non-specific binding sites and permeabilize cells for 30 min at 4°C and specific binding sites were stained with mouse anti-HRV-A16 antibody (1:200; QED Bioscience, San Diego, CA, USA), anti-Mucin 5AC antibody (1:200; Thermo Fisher Scientific) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells

doi: 10.3389/fcimb.2020.00278

Figure Lengend Snippet: Effects of epithelial infection by HRV-A16 on its receptor and epithelial cell markers ALI-PBEC were infected with HRV-A16 (MOI 0.1, 1, 5) for 1 h and were incubated for 24, 48, or 72 h. (A–E) The replication of HRV-A16 vRNA and gene expression of ICAM-1, MUC5AC, SCGB1A1, and FOXJ1 were measured by qPCR. Data are shown as target gene expression normalized for RPL13A and ATP5B. Data are mean values ± SEM. n = 8 independent donors. Analysis of differences was conducted using two-way ANOVA with a Tukey post-hoc test. Significant differences are indicated by * P < 0.05 compared with control. (F) At 48 h after HRV-A16 infection, cells were fixed in 4% paraformaldehyde solution and stained using immunofluorescence with primary antibodies against MUC5AC (goblet cell markers, green) or acetylated α-tubulin and FOXJ1 (ciliated cell markers, green, and red) in combination with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining (blue). Images shown are representative for results obtained with cells from four different donors with 630 x original magnification ( n = 4).

Article Snippet: PBS/1% (w/v) BSA/0.3% (w/v) Triton-X-100 (PBT) was used to block non-specific binding sites and permeabilize cells for 30 min at 4°C and specific binding sites were stained with mouse anti-HRV-A16 antibody (1:200; QED Bioscience, San Diego, CA, USA), anti-Mucin 5AC antibody (1:200; Thermo Fisher Scientific) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Infection, Incubation, Gene Expression, Targeted Gene Expression, Control, Staining, Immunofluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells

doi: 10.3389/fcimb.2020.00278

Figure Lengend Snippet: The target of HRV-A16 in human bronchial epithelial cells. (A) At 8 h after HRV-A16 infection, cells were fixed in 4% paraformaldehyde solution and stained using immunofluorescence with mouse anti-HRV-A16 antibody (green), primary antibodies against FOXJ1 (ciliated cell marker, red) in combination with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining (blue). Images shown are representative for results obtained with cells from four different donors with 630 x original magnification ( n = 4). (B) PBEC were differentiated for 14 days in the presence of either 5 uM DAPT or solvent control. On day 14, cells were infected by HRV-A16 (MOI 1). After infection, the basal medium was replaced by fresh medium contain 5 uM DAPT or solvent control. Cultures were incubated for 24 h before harvesting lysis of cells for isolation of RNA. The vRNA and gene expression of cell markers (MUC5AC, SCGB1A1, TP63, and FOXJ1) were measured by qPCR. Data are mean values ± SEM. Data are shown as target gene expression normalized for RPL13A and ATP5B. n = 4 independent donors. Analysis of differences was conducted using paired t -test and one-way ANOVA with a Turkey post-hoc test. Significant differences are indicated by * P < 0.01 compared with control or HRV-A16.

Article Snippet: PBS/1% (w/v) BSA/0.3% (w/v) Triton-X-100 (PBT) was used to block non-specific binding sites and permeabilize cells for 30 min at 4°C and specific binding sites were stained with mouse anti-HRV-A16 antibody (1:200; QED Bioscience, San Diego, CA, USA), anti-Mucin 5AC antibody (1:200; Thermo Fisher Scientific) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Infection, Staining, Immunofluorescence, Marker, Solvent, Control, Incubation, Lysis, Isolation, Gene Expression, Targeted Gene Expression

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells

doi: 10.3389/fcimb.2020.00278

Figure Lengend Snippet: Modulation of a network of genes relating to SPDEF by HRV-A16. ALI-PBEC were infected with HRV-A16 (MOI 0.1, 1, 5). Cells were harvested at 48 h after infection. (A–D) The gene expressions of SPDEF, FOXA3, AGR, and FOXA2 were measured by real-time PCR Data are shown as target gene expression normalized for RPL13A and ATP5B. Data are mean values ± SEM. n = 8 independent donors. Analysis of differences was conducted by paired one-way ANOVA with a Tukey post-hoc test. Significant differences are indicated by * P < 0.05 compared with control.

Article Snippet: PBS/1% (w/v) BSA/0.3% (w/v) Triton-X-100 (PBT) was used to block non-specific binding sites and permeabilize cells for 30 min at 4°C and specific binding sites were stained with mouse anti-HRV-A16 antibody (1:200; QED Bioscience, San Diego, CA, USA), anti-Mucin 5AC antibody (1:200; Thermo Fisher Scientific) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Infection, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells

doi: 10.3389/fcimb.2020.00278

Figure Lengend Snippet: Effects of tiotropium bromide and fluticasone propionate on viral infection in ALI-PBEC. ALI-PBEC were pre-treated with tiotropium or fluticasone and infected with HRV-A16 (MOI 5). Cells were harvested at 48 h after infection. (A,B) The vRNA expression of HRV-A16 ( n = 7) and HRV-1B ( n = 3) together with (C,D) viral particles were measured by TCID50 assay ( n = 8 or 3). (E,F) Gene expression of ICAM-1 ( n = 7) and LDLR ( n = 3) were examined by qPCR. Data are shown as target gene expression normalized for RPL13A and ATP5B or TCID50/mL. Data are mean values ± SEM. n = 3, 7, and 8 independent donors. Analysis of differences was conducted by two-way ANOVA with a Tukey post-hoc test Significant differences are indicated by * P < 0.05 compared with control or HRV-A16 group.

Article Snippet: PBS/1% (w/v) BSA/0.3% (w/v) Triton-X-100 (PBT) was used to block non-specific binding sites and permeabilize cells for 30 min at 4°C and specific binding sites were stained with mouse anti-HRV-A16 antibody (1:200; QED Bioscience, San Diego, CA, USA), anti-Mucin 5AC antibody (1:200; Thermo Fisher Scientific) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Infection, Expressing, TCID50 Assay, Gene Expression, Targeted Gene Expression, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells

doi: 10.3389/fcimb.2020.00278

Figure Lengend Snippet: Effects of tiotropium and fluticasone on expression of MUC5AC and MUC5B in ALI-PBEC. ALI-PBEC were pre-treated with tiotropium or fluticasone and infected with HRV-A16 (MOI 5). Cells were harvested at 48 h after infection. The gene expression of MUC5AC (A,C) and MUC5B (B,D) were measured by real-time PCR. Data are mean values ± SEM. n = 10 or 6 independent donors. Data are shown as target gene expression normalized for RPL13A and ATP5B. Analysis of differences was conducted by two-way ANOVA with a Tukey post-hoc test Significant differences are indicated by * P < 0.05 compared with control or HRV-A16 group.

Article Snippet: PBS/1% (w/v) BSA/0.3% (w/v) Triton-X-100 (PBT) was used to block non-specific binding sites and permeabilize cells for 30 min at 4°C and specific binding sites were stained with mouse anti-HRV-A16 antibody (1:200; QED Bioscience, San Diego, CA, USA), anti-Mucin 5AC antibody (1:200; Thermo Fisher Scientific) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Expressing, Infection, Gene Expression, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells

doi: 10.3389/fcimb.2020.00278

Figure Lengend Snippet: Effects of tiotropium and fluticasone on mucin release in ALI-PBEC. ALI-PBEC were pre-treated with tiotropium or fluticasone and infected with HRV-A16 (MOI 5). Cells were harvested at 48 h after infection. The protein levels of Mucin 5AC (A,C) and Mucin 5B (B,D) were measured by ELISA. n = 10 or 6 independent donors. (E) Mucin 5AC positive cells were quantified as positive cell numbers/mm (length of basal membrane, n = 4). Data are mean values ± SEM and shown as arbitrary units/mL according to standard line or MUC5AC+ cells/mm. Analysis of differences was conducted by paired one-way or two-way ANOVA with a Tukey post-hoc test Significant differences are indicated by * P < 0.05 compared with control or HRV-A16 group.

Article Snippet: PBS/1% (w/v) BSA/0.3% (w/v) Triton-X-100 (PBT) was used to block non-specific binding sites and permeabilize cells for 30 min at 4°C and specific binding sites were stained with mouse anti-HRV-A16 antibody (1:200; QED Bioscience, San Diego, CA, USA), anti-Mucin 5AC antibody (1:200; Thermo Fisher Scientific) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Membrane, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells

doi: 10.3389/fcimb.2020.00278

Figure Lengend Snippet: Modulation of fluticasone on SPDEF-regulated genes in ALI-PBEC. ALI-PBEC were pre-treated with tiotropium or fluticasone and infected with HRV-A16 (MOI 5). Cells were harvested at 48 h after infection. The gene expression of SPDEF (A,B) , FOXA3 (C,D) and AGR2 (E,F) were measured by real-time PCR. Data are shown as target gene expression normalized for RPL13A and ATP5B. Data are mean values ± SEM. n = 10 or 6 independent donors. Analysis of differences was conducted by two-way ANOVA with a Tukey post-hoc test Significant differences are indicated by * P < 0.05 compared with control or HRV-A16 group.

Article Snippet: PBS/1% (w/v) BSA/0.3% (w/v) Triton-X-100 (PBT) was used to block non-specific binding sites and permeabilize cells for 30 min at 4°C and specific binding sites were stained with mouse anti-HRV-A16 antibody (1:200; QED Bioscience, San Diego, CA, USA), anti-Mucin 5AC antibody (1:200; Thermo Fisher Scientific) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Infection, Gene Expression, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells

doi: 10.3389/fcimb.2020.00278

Figure Lengend Snippet: Inhibition of fluticasone on ATP-induced mucin production and involvement of extracellular ATP in HRV-induced mucin production and release in ALI-PBEC. (A) ATP (100 μM) treated cells in the presence of tiotropium or fluticasone. Cells were incubated for 24 h. The gene expression of MUC5AC was measured using qPCR. (B) ALI-PBEC were infected by HRV-A16 (MOI 5) after pre-treatment with suramin (10 μM). Cells were incubated for 48 h after infection. The gene expression of MUC5AC was measured by qPCR. (C) Levels of Mucin 5AC were assessed by ELISA assay. Gene expression were normalized for RPL13A and ATP5B. Data are shown as normalized or fold expression. Data are mean values ± SEM. n = 4 independent donors. Analysis of differences was conducted with paired one-way or two-way ANOVA with a Tukey post-hoc test Significant differences are indicated by * P < 0.05 compared with control, HRV-A16 and ATP group.

Article Snippet: PBS/1% (w/v) BSA/0.3% (w/v) Triton-X-100 (PBT) was used to block non-specific binding sites and permeabilize cells for 30 min at 4°C and specific binding sites were stained with mouse anti-HRV-A16 antibody (1:200; QED Bioscience, San Diego, CA, USA), anti-Mucin 5AC antibody (1:200; Thermo Fisher Scientific) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Inhibition, Incubation, Gene Expression, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Control