Figure 5

Live/Dead assays determined calcium dependency and extracellular signal-regulated kinase (ERK) contribution to cell death induced by pH 6.0 with IL-1β. The number of dead fibroblast-like synoviocytes (FLS) and the number of live FLS were counted to obtain the percent dead. (A - F) Representative images from FLS stained using a Live/Dead Viability/Cytotoxicity kit. Live cells were stained with calcein AM (green), dead cells were stained with ethidium homodimer (red). ( A and B ) Show images of FLS treated by pH 7.4 without or with IL-1β, respectively. ( C and D ) Show images of FLS treated by pH 6.0 and IL-1β without or with intracellular calcium chelating agent (BAPTA, 1 μM), respectively; ( E and F ) show images of FLS treated by pH 6.0 and IL-1β without or with inhibitor of ERK (PD98059,10 μM), respectively. (G) Quantitative analysis shows approximately 30% dead FLS after treatment with pH 6.0 and IL-1β, which was significantly greater than FLS treated with pH 6.0 and IL-1β with BAPTA, and pH 7.4 and IL-1β with and without BAPTA (*P <0.01). (H) Quantitative analysis shows approximately 30% dead FLS after treatment with pH 6.0 and IL-1β, which was significantly greater than FLS treated with pH 6.0 and IL-1β with PD98059 (10 μM), and FLS with or without IL-1β and pH 7.4 (*P <0.001). Data are mean ± standard error of the mean.