Figure 3

Analysis of mitochondrial membrane potential (Δψm) after diosgenin treatment. Human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were cultured in 10% FCS medium for 48 hours and then treated (b) or not (a) with 40 μM diosgenin. Δψm was analyzed in adherent RA FLS after 24 hours of treatment, using the potential-dependent aggregate-forming lipophilic cation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide). Red fluorescence (a) represents mitochondria with intact membrane potential whereas green fluorescence (b) represents de-energized mitochondria. Staining with DAPI (4',6-diamidino-2-phenylindole), showed that diosgenin treatment of cells altered the extracellular and nuclear membrane permeability, as is shown by the nuclear localization of the DAPI (b, white arrows) in comparison with untreated cells (a). Pictures were taken with a Nikon microscope ECLIPSE E800 (original magnification ×400). One of three representative experiments from three different patients is shown.