FIG. 6.

MIP-3α activity in keratinocytes requires NF-κB signaling. Keratinocytes (1 × 10⁵) were seeded in six-well dishes and transfected 24 h later with either 100 ng MIP-3αWT (A) or MIP-3αmNFκB (B) by use of Fugene 6. At 24 h after transfection, cells were washed with 1× PBS and treated with media alone or media containing 10 μg/ml Poly(I) · Poly(C), 10 ng/ml TNF-α, or 10 ng/ml IL-1β for 6 h. Following treatment, cells were harvested, lysed, and measured for luciferase activity. Results are expressed as the mean numbers of relative luciferase units ± SD of two independent experiments. (C and D) Primary human foreskin keratinocytes stably expressing vector alone (Babe), HPV-16 E6/E7 (E6/E7), HPV-16 E6/E7stop (E6), HPV-16 E6(C66G/C136G)/E7stop [E6(C66G/C136G)], HPV-16 E6stop/E7 (E7), or HPV-16 E6stop/E7.2 (E7.2) were treated with 100 μg/ml Poly(I) · Poly(C) for 16 h. Following treatment, cells were washed and refed with minimal EpiLife for 4 h. RNA was harvested from the cells, and mRNA levels for MIP-3α were determined by real-time qPCR. Results are expressed as the means ± SD of three experiments using two independent cell lines (* P < 0.01).