Journal: Scientific Reports
Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis
Figure Lengend Snippet: IAV-induced expression of Runx3 is primarily mediated by MDA5−NF-κB pathway. ( a , b ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con), human MDA5 siRNA-2 (M), TLR3 siRNA-2 (T), both MDA5 and TLR3 siRNAs (M + T), RIG-1 siRNA-1 (S1), or RIG-1 siRNA-2 (S2), grown for 72 h then infected without (−) or with (+) H1N1 PR/8/34 at MOI of 1 for 24 h. ( c,d ) BEAS-2B cells were transfected with control H 2 O (Con), or different amounts of RNA isolated from uninfected BEAS-2B cells (C-RNA) or H1N1-infected BEAS-2B cells (V-RNA) treated with or without calf intestine alkaline phosphatase (CIAP). After 24 h, IFNβ released into the medium was measured by ELISA ( c ) or equal amounts of cell lysates were subjected to Western blot analysis ( d ). ( e )BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con) or human MDA5 siRNA-2 (M), grown for 72 h, then transfected with control H 2 O (Control) or total RNA isolated from H1N1-infected BEAS-2B cells (V-RNA-1, 300 ng; V-RNA-2, 900 ng)) for 24 h. ( f ) BEAS-2B cells were infected without (−) or with H1N1 at MOI of 1 and then incubated with DMSO, piceatannol (Piceat, 15 μM), BAY11-7082 (BAY, 10 μM), SC514 (8 μM), or Amlexanol (Amlex, 10 μM) for 24 h. ( g ) BEAS-2B cells were transfected with 20 nM non-targeting control siRNA (Con), human p65, IKKβ siRNA-1 (β-1) or IKKβ siRNA-2 (β-2), grown for 72 h, then infected with H1N1 at MOI of 1 for 24 h. ( h ) Primary signal pathway for Runx3 induction by IAV H1N1. ( i ) BEAS-2B cells were treated with control PBS, IFNα (300 U/ml), IFNγ (200 ng/ml), or IFNλ (200 ng/ml) for 24 h. Equal amounts of cell lysates from ( a–i ) were subjected to Western blot analysis with indicated antibodies. Results represent Western blots of three independent experiments. Relative changes in protein levels of Runx3 of three independent experiments were measured by densitometric analysis using ImageJ 1.47 software and normalized to actin and are represented as percentage of control siRNA (n = 3).
Article Snippet: Antibodies and reagents Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology.
Techniques: Expressing, Transfection, Infection, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Software