Journal:

Article Title: Upregulation of the TLR3 Pathway by Kaposi's Sarcoma-Associated Herpesvirus during Primary Infection ▿

doi: 10.1128/JVI.02590-07

Figure Lengend Snippet: siRNA against TLR3 diminishes the induction of CXCL10, IFN-β1, and IRF-1 gene expression from KSHV-infected THP-1 cells. (A) Western blot of TLR3 protein levels in KSHV-infected or mock-infected THP-1 cells. (B) THP-1 cells were nucleofected with a control luciferase siRNA plasmid, psiLUC (lane 1), or a TLR3-specific siRNA plasmid, psiTLR3 (lanes 2 [3 μg psiTLR3] and 3 [8 μg psiTLR3]), followed by KSHV infection and Western blotting for TLR3. (C) THP-1 cells were nucleofected with 3 μg control siRNA or a TLR3-specific siRNA plasmid, psiTLR3. Real-time PCR profiling of TLR pathway genes indicated a significant reduction in the increase of mRNA upon pretreatment of THP-1 cells with the siRNA specific to TLR3, indicating that these downstream genes are specifically activated through TLR3 in response to KSHV infection. (D) THP-1 cells were nucleofected with 3 μg control siRNA (siLUC) or TLR3-specific siRNA (psiTLR3). Cells were infected with KSHV, and CXCL10 was measured 16 h postinfection. The CXCL10 ELISA shows a 70% decrease in CXCL10 secretion from KSHV-infected THP-1 cells transfected with psiTLR3 compared to cells transfected with psiLUC. (E) THP-1 cells were transfected with 3 μg of psiTLR3 or psiLUC. At 48 h posttransfection, cells were treated with poly(I:C) for 24 h. RNA was harvested and subjected to RT-PCR using TLR3 and CXCLIO primers.

Article Snippet: The SuperArray TLR PCR array (APH-018) was used to analyze the cDNA obtained from infected cells for the activation of the TLRs or any of the downstream genes in each of the pathways.

Techniques: Gene Expression, Infection, Western Blot, Control, Luciferase, Plasmid Preparation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Reverse Transcription Polymerase Chain Reaction