Effects of secreted TNF-α in supernatant stimulated pro-inflammatory cytokines (IL-1β and IL-8) expression and effects of membrane TNF-α on IL-8 expression in HaCaT cells.

<p>HaCaT-TNF-α cells were treated with 1.0 <b>μ</b>g/mL doxycycline (Dox) for 48 hrs. Supernatant collected was serially diluted with fresh medium in the ratio (supernatant fresh medium) 1:1.5, 1:2, 1:3 and 1:4. HaCaT cells were incubated with diluted supernatant for 24 hrs and subsequently treated with 10 ng/mL IFN-γ for a further 24 hrs. Expression levels of IL-1β and IL-8 were quantitated by qRT-PCR. Relative expression of IL-1β (A) and IL-8 (B) are shown. (C) HaCaT-TNF-α cells were cultured at different seeding densities and treated with 1 <b>μ</b>g/ml Dox for 48 hr. Cells harvested at approximately 50% or 90% confluence. IL-8 gene expression from cells at 50% and 90% confluency were analyzed by RT qPCR. Relative expression of IL-8 are shown. Results are represented as the Mean ± SEM from three independent experiments compared with control untreated *(<i>p</i><0.05), **(<i>p</i><0.01).