Figure 6

Immunocytochemical detection of gliostatin (GLS) in fibroblast-like synoviocytes. Fibroblast-like synoviocytes were stimulated by the presence or absence of 300 nM mithramycin for 30 minutes, followed by further incubation with or without tumor necrosis factor-alpha (TNF-α) (1 ng/mL) for 24 hours, and were immunostained with GLS antibody (red). Cell nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar represents 50 μm (A). Total red intensity of immunostaining in a random field was quantified by ImageJ. The numbers of cells were counted in the field. Data (intensity/cell) were presented as mean ± standard error of the mean (SEM) of nine determinations. Statistical significance was calculated by using the Mann-Whitney U test: compared with controls *P < 0.05, compared with samples with TNF-α alone ^†P < 0.05 (B). MIT, mithramycin.