Figure 3.

Effect of tumor necrosis factor alpha (TNFα) on the expression of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) in rheumatoid arthritis (RA) fibroblast-like synovial cells (FLS). (A) RA FLS were treated with or without 10 ng/ml TNFα for 24 hours, and M-CSF and IL-34 mRNA levels were determined in cell lysates by quantitative reverse transcriptase-PCR (qRT-PCR). The relative expression of M-CSF (left) and IL-34 (right) mRNA after TNFα treatment is presented as fold-change in gene expression compared to untreated controls (none). Bars indicate the mean and SD of M-CSF or IL-34 mRNA expression relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (*P< 0.05, N.S., not significant, compared to none). (B) Cells were cultured as described in panel A, and the concentration of M-CSF (left) or IL-34 (right) in the medium was measured by an enzyme-linked immunosorbent assay (ELISA). (C) TNFα-mediated induction of M-CSF (left) or IL-34 (right) protein production was converted into fold change relative to that of control (none). Bars indicate the mean and SD of M-CSF or IL-34 protein relative to that of control (*P < 0.05, N.S., not significant, compared to none). (D) Nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) dependent IL-34 mRNA expression upon TNFα stimulation in RA FLS. RA FLS were pretreated with the vehicle (dimethyl sulfoxide, 10 μM), NF-κB inhibitor, pyrrolidine dithiocarbamate(10 μM), the JNK inhibitor, SP600125 (10 μM), the p38 inhibitor, SB203580 (10 μM), or the extracellular signal-regulated protein kinase (ERK) inhibitor, PD98059 (10 μM), for 30 minutes and then treated with 10 ng/ml TNFα for 24 hours. Untreated RA FLS were used as a control (none). M-CSF and IL-34 mRNA levels were determined in cell lysates by qRT-PCR. The relative expression of IL-34 mRNA is presented as fold change in gene expression compared to untreated vehicle controls (*P < 0.05, N.S., not significant, compared to veh). none, no treatment; veh, vehicle; SP, SP600125; SB, SB203580; PD, PD98059.

Hwang et al. Arthritis Research & Therapy 2012 14:R14   doi:10.1186/ar3693
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