Cyp7b activity and mRNA expression is inhibited by SN50 in fibroblast-like synoviocytes. (a) Human fibroblast-like synoviocytes (FLS; SCRO.14.SF, passages 10–12) were plated at 1 × 105 cells/well in a 24-well plate and preincubated in the presence or absence (-) of the SN50 inhibitor for 2 hours. Thereafter, the cells were incubated with (solid bars) or without (open bars) tumour necrosis factor (TNF)-α for another 24 hours with 1.5 × 10-8 mol/l 3H-dehydroepiandrosterone (DHEA). The formation of [3H]-7α-hydroxy-dehydroepiandrosterone (7α-OH-DHEA) from [3H]-DHEA, representing cytochrome p450 enzyme 7b (Cyp7b) activity, was determined by high-performance liquid chromatography. The amount of 7α-OH-DHEA is expressed as the percentage [3H]-7α-OH-DHEA of the total amount of [3H]-label measured. Results are expressed as the mean ± standard error of the mean of triplicate samples. The data are representative of two independent experiments. *P < 0.005 (Student's t-test). (b) Human FLS (1 × 105 cells/well) were isolated from five different rheumatoid arthritis patient biopsies. Cells (1 × 105/well) were incubated in the presence and absence of TNF-α and in the presence of SN50 for 2 hours, as described in Materials and methods. Results are representative for one of the two independent experiments. SCRO.12.SF passage 2, SCRO.11.SF passage 3, SCRO.03.SF passage 8, SCRO.01.SF passage 6 and SCRO.08.SF passage 4 were used. *P < 0.005 versus TNF-α (Student's t-test). (c) FLS (SCRO.14.SF; passages 10–12) were incubated for 6 hours with 0.5 ng/ml TNF-α, SN50 200 μg/ml plus 0.5 ng/ml TNF-α, or incubated with medium control (-). Reverse transcription PCR was done using GAPDH and Cyp7b specific primers (35 cycles). The data are representative of two independent experiments. (d) FLS fibroblasts (SCRO.14.SF; passages 10–12) were grown on chamber slides. Cells were incubated for 2 hours in the presence or absence of 200 μg/ml SN50 before incubation for 30 min in the presence or absence of TNF-α (0.5 ng/ml). Immunoperoxidase staining was carried out with an antibody against nuclear factor-κB (NF-κB)p65 conjugated to peroxidase. Data are representative for three independent experiments.