Figure 1. Extracellular GrK is not cytotoxic. HFLs were serum starved and incubated with growth media in the presence or absence of GrK (100 nM, 200 nM) for 24 h and cell viability was assessed using the WST-1 assay. All data are expressed as mean absorbance 6 SEM from three independent triplicate experiments. doi:10.1371/journal.pone.0021484.g001 Granzyme K Activates PAR-1 viability and proliferation in HFLs. Cell viability was measured using the WST-1 assay (Roche, Ltd. Missasauga, ON, Canada) according to manufacturer’s instructions. Here, 96-well culture dishes were seeded with 16104 cells and incubated with media alone or GrK (100 nM, 200 nM) for 24 h. Cells were then incubated with 1:10 dilution of WST-1 reagent for 3 h and absorbance was read at 450 nM. Cell proliferation was assessed using cell counts. Cells were seeded in 12-well culture dishes and incubated with varying concentrations of GrK for 48 h, with or without ATAP-2 (5 mg/ml). Cells were then trypsinized, centri- fuged for 5 min 1006 g, re-suspended in 1 ml PBS, diluted 1:1 in 0.4% Trypan Blue and counted using a hemocytometer. Cells treated with ATAP-2 were incubated for 30 min prior to treatment with GrK. Statistical analysis All data are expressed as mean +/2 SEM for three separate experiments. Statistical analysis was performed using One-Way ANOVA followed by Dunnets post hoc analysis for multiple group comparisons. Differences were considered significant at p,0.05. Results Extracellular GrK is not cytotoxic As extracellular GrA and GrB may induce cell death through the cleavage of extracellular matrix proteins and the subsequent induction of anoikis, we examined the impact of extracellular GrK Figure 2. GrK stimulates IL-6, IL-8 and MCP-1 protein production in lung fibroblasts. (A–C) HFLs were exposed to concentrations of GrK ranging from 10–300 nM at for 24 h. Cell culture supernatants were then collected and analyzed for IL-6, IL-8 and MCP-1 production by ELISA. Cell counts were used to normalize cytokine concentrations by controlling for variability in cell numbers. Control wells include cells treated with media alone or cells treated with heat in-activated GrK (100 nM). All data are expressed as mean protein production (pg/106 cells) 6 SEM from three independent triplicate experiments. * p,0.05 when compared to media alone; ** p,0.01 when compared to media alone. doi:10.1371/journal.pone.0021484.g002 PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21484