IL-27 enhances TLR3 expression in human melanomas, which could account for the cooperative effect between IL-27 and poly(I:C).

<p>(A-C) Human melanoma cell lines (SK-MEL-13, -28, and -37) were stimulated with IL-27 (1, 3, 10 ng/ml) and/or poly(I:C) (1 µg/ml) for 24 h or the indicated times. Total RNA was then extracted and subjected to RT-PCR analysis. Similar results were obtained in more than two independent experiments. (D and E) Cell lysate was also prepared after the stimulation for 48 h and subjected to Western blotting with antibodies against TLR3, RIG-I, MDA5 and β-actin. Similar results were obtained in more than two independent experiments. (F) SK-MEL-37 cells were transfected with siRNA specific to TLR3 or control siRNA for 24 h. These cells were then stimulated with IL-27 (10 ng/ml) and poly(I:C) (1 µg/ml) for a further 24 h, and total cell lysate was prepared and subjected to Western blot using anti-TLR3 and anti-β-actin. (G) The siRNA-transfected cells were also stimulated with IL-27 (10 ng/ml) and poly(I:C) (1 µg/ml) for a further 48 h and pulsed with ³H-thymidine for the last 8 h in triplicate. ³H-thymidine incorporation was measured, and relative proliferation (%) to that of respective unstimulated cells was calculated. Data are shown as means ± SD. * indicates <i>p</i> < 0.05 compared with no siRNA and control siRNA. Similar results were obtained in two independent experiments.