Fig. 2.

Cigarette smoke condensate (CSC) increases expression of Tnf-α in mouse middle ear epithelial cell line (mMEEC). (A) Real-time reverse transcriptase-polymerase chain reaction analysis showed marked increased expression of Tnf-α mRNA with 20 and 40 μg/mL CSC at 8 hours. (B) Diagram of the proximal −1332 bp of the Tnf-α 5′ flanking region with putative NF-κB sites depicted. This region was cloned onto a PGL3 luciferase vector for reporter assay experiments. (C) Luciferase reporter assays with TnfαLuc demonstrated statistically significant increases in Tnf-α promoter activity with CSC. The results are normalized to β-Gal cotransfection to account for cell number and transfection efficiency.