Figure 4
Figure 4. Defective expression of TLR2 resulted in abrogation of HSV-1–induced IL-15 gene expression. (A) THP-1 cells were transfected with Sp1 specific siRNA or its control siRNA. After 48 hours, the cell lysates were examined for Sp1 protein expression by immunoblotting. A representative Western blot is shown. (B) Sp1 siRNA-transfected THP1 cells (top panel) and THP1 cells pretreated with mithramycin, WP631, or DMSO as control (bottom panel) were assessed for their expression of TLR2. THP-1 surface expression of TLR2 was determined by flow cytometry as described in “Flow cytometry.” (C) THP-1 cells subjected to the different treatments (as indicated) were incubated with mock or HSV-1. Cells were harvested 6 hours after incubation, and total RNA was analyzed for IL-15 gene expression by RT-PCR. The figure shows IL-15 mRNA (top panel) and 18S rRNA (bottom panel) and is representative of 3 independent experiments. (D) Similarly, real-time RT-PCR was performed and data are expressed as the ratio of IL-15 mRNA in treated samples to the untreated sample (after normalization with reference dye and housekeeping gene). *Observed difference is statistically significant (P < .001) comparing gene expression with wild-type cells exposed to HSV-1.

Defective expression of TLR2 resulted in abrogation of HSV-1–induced IL-15 gene expression. (A) THP-1 cells were transfected with Sp1 specific siRNA or its control siRNA. After 48 hours, the cell lysates were examined for Sp1 protein expression by immunoblotting. A representative Western blot is shown. (B) Sp1 siRNA-transfected THP1 cells (top panel) and THP1 cells pretreated with mithramycin, WP631, or DMSO as control (bottom panel) were assessed for their expression of TLR2. THP-1 surface expression of TLR2 was determined by flow cytometry as described in “Flow cytometry.” (C) THP-1 cells subjected to the different treatments (as indicated) were incubated with mock or HSV-1. Cells were harvested 6 hours after incubation, and total RNA was analyzed for IL-15 gene expression by RT-PCR. The figure shows IL-15 mRNA (top panel) and 18S rRNA (bottom panel) and is representative of 3 independent experiments. (D) Similarly, real-time RT-PCR was performed and data are expressed as the ratio of IL-15 mRNA in treated samples to the untreated sample (after normalization with reference dye and housekeeping gene). *Observed difference is statistically significant (P < .001) comparing gene expression with wild-type cells exposed to HSV-1.

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