HMGB-1 induces IL-8 mRNA expression and NF-κB activation via PAR-2 receptors by keratinocytes. (A) IL-8 mRNA levels were evaluated by qRT-PCR on PAR2- or control siRNA–transfected keratinocytes (HaCaT cell line) exposed to Karpas-299 (Karpas) and SU-DHL-1 cell line (ALK⁺ALCL) supernatants for 24 hours. The level of IL-8 mRNA was normalized to that from HaCaT cells cultured in DMEM (control medium). (B) Protein lysates from HaCaT cells exposed or not to ALK⁺ALCL cell line supernatants were subjected to western-blotting with anti–NF-κB p65 and anti–phospho-Ser⁵³⁶ NF-κB p65 Abs. β-actin was used as a loading control. A vertical line has been inserted to indicate a repositioned gel lane. (C-D) The same experiments as in panels A and B were performed on PAR2- or control siRNA–transfected HaCaT cells treated with rhHMGB-1 or not (PBS). IL-8 mRNA levels were normalized to the endogenous housekeeping gene GAPDH. Results are expressed as the fold increase compared with the control PBS condition (expression level = 1), and represent the mean ± SD of 3 independent experiments done in triplicate; *P < .05 by Student t test (C). Lysates from HaCaT cells were subjected to Western blotting with anti–NF-κB p65 and anti–phospho-Ser⁵³⁶ NF-κB p65 Abs. β-actin was used as a loading control (D).