m50 super 8× topflash reporter plasmid  (Addgene inc)

Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: RSV induces β-catenin during infection of lung epithelial cells. (A) Human lung epithelial A549 cells were infected with RSV (MOI = 1) for 0–16 h. β-catenin and actin levels were determined in cell lysates by western blotting using corresponding antibodies. (B) Densitometry analysis of β-catenin protein levels relative to actin protein (β-catenin/Actin) in RSV-infected A549 cells. (C) TOP-Flash luciferase assay of A549 cells co-transfected with firefly-luciferase-TOP-Flash and renilla-luciferase plasmids. Co-transfected cells were infected with RSV (MOI = 1) for 16 h. A dual luciferase reagent was utilized to determine firefly and renilla luciferase activity. The relative TOP-Flash luciferase activity was calculated based on the mean value of firefly/renilla luciferase activity. The value is represented as a fold change in TOP-Flash activity in RSV-infected cells compared to mock infected cells. The densitometric values represent the mean ± SEM from three independent studies (* p ≤ 0.05). Luciferase assay represents mean ± SEM from three independent experiments performed in triplicates [* p ≤ 0.05 ( n = 24; technical replicates)].

Article Snippet: Cells (A549, FL-catenin, and Δ-catenin cells) were co-transfected with M50 Super 8× TOP-Flash firefly luciferase reporter plasmid (Addgene) and a plasmid encoding Renilla luciferase for 16 h. Transfected cells were either infected with RSV or treated with purified HBD3 protein.

Techniques: Infection, Western Blot, Luciferase, Transfection, Activity Assay

Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: Reduced pro-inflammatory response during RSV infection of lung epithelial cells lacking full-length β-catenin protein. (A) CRISPR-Cas9 technology was used to generate stable human lung epithelial A549 cells lacking full-length β-catenin protein. Cell lysates from A549 cells expressing full-length 92 kDa β-catenin protein (FL-catenin-A549 cells) and A549 cells expressing truncated 82 kDa β-catenin protein (Δ-catenin-A549 cells) were subjected to western blotting with β-catenin antibody. (B) TOP-Flash luciferase assay of A549 cells expressing either FL-catenin or Δ-catenin were co-transfected with firefly-luciferase-TOP-Flash and renilla-luciferase plasmids. Co-transfected cells were treated with Lithium Chloride (LiCl; 25 mM) for 24 h. A dual luciferase reagent was utilized to determine firefly and renilla luciferase activity. The relative TOP-Flash luciferase activity was calculated based on the mean value of firefly/renilla luciferase activity. The value is represented as a fold change in TOP-Flash activity in LiCl-treated cells compared to vehicle (water) treated cells. (C) A549 cells expressing either FL-catenin or Δ-catenin were infected with RSV (MOI = 3). Medium supernatant collected from these cells was analyzed for IL-8 production by ELISA. Luciferase assay represents mean ± SEM from two independent experiments performed in triplicates [* p ≤ 0.05 ( n = 16; technical replicates)]. ELISA data are shown as Mean ± SEM [* p ≤ 0.05 ( n = 24; technical replicates; three independent experiments)].

Article Snippet: Cells (A549, FL-catenin, and Δ-catenin cells) were co-transfected with M50 Super 8× TOP-Flash firefly luciferase reporter plasmid (Addgene) and a plasmid encoding Renilla luciferase for 16 h. Transfected cells were either infected with RSV or treated with purified HBD3 protein.

Techniques: Infection, CRISPR, Expressing, Western Blot, Luciferase, Transfection, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5

doi: 10.3389/fmicb.2023.1186510

Figure Lengend Snippet: Human defensin-3 induces β-catenin activity in lung epithelial cells. (A) Human lung epithelial A549 cells were treated with either vehicle (0.1% BSA in PBS) or purified human defensin-3 (HBD3) protein (10 μg/ml) for 16 h. β-catenin and actin levels were determined in cell lysates by western blotting using corresponding antibodies. (B) Densitometry analysis of β-catenin protein levels relative to actin protein (β-catenin/Actin) in the vehicle and HBD3-treated A549 cells. (C) TOP-Flash luciferase assay of A549 cells co-transfected with firefly-luciferase-TOP-Flash and renilla-luciferase plasmids. Co-transfected cells were treated with either vehicle (0.1% BSA in PBS) or HBD3 for 16 h. A dual luciferase reagent was utilized to determine firefly and renilla luciferase activity. The relative TOP-Flash luciferase activity was calculated based on the mean value of firefly/renilla luciferase activity. The value is represented as a fold change in TOP-Flash activity in HBD3-treated cells compared to vehicle-treated cells. The densitometric values represent the mean ± SEM from three independent studies (* p ≤ 0.05). Luciferase assay represents mean ± SEM from two independent experiments performed in triplicates [* p ≤ 0.05 ( n = 6; technical replicates)].

Article Snippet: Cells (A549, FL-catenin, and Δ-catenin cells) were co-transfected with M50 Super 8× TOP-Flash firefly luciferase reporter plasmid (Addgene) and a plasmid encoding Renilla luciferase for 16 h. Transfected cells were either infected with RSV or treated with purified HBD3 protein.

Techniques: Activity Assay, Purification, Western Blot, Luciferase, Transfection