Fig. 5.
Down-regulation of FADD-like interleukin (IL)-1β-converting enzyme (FLICE) inhibitory protein, (FLIP) in the synovial fibroblasts by hydroxychloroquine (HCQ). (a) Dose-dependent reduction of FLIP mRNA expression by HCQ. Fibroblast-like synoviocytes (FLS) were incubated without (lane 1) or with 1, 10 and 100 µM of HCQ for 4 h (lane 2–4), and then analysed for FLIPL (the long form of FLIP) mRNA expression by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR). Amplification of the housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), was used as the control. The mRNA levels are expressed as the fold increase relative to the mRNA from the cells treated with 100 µM of HCQ (lane 4), and were corrected for the levels of the GAPDH mRNA signal. The data represent the results from one of three similar experiments. (b) Time-dependent decrease in FLIP protein expression by HCQ. The FLS were treated without (lane 1) or with HCQ 100 µM for 1–6 h (lane 2–4), and then examined for FLIP protein expression by Western blotting analysis using anti-human FLIP monoclonal antibodies (mAb), which recognizes both FLIPL (55 kDa) and FLIPS (27/28 kDa) isoforms. The data are a representative result of three independent experiments. (c) Time kinetics analysis of FLIPL protein expression (solid circle) in the HCQ (100 µM)-treated FLS and the extent of apoptosis (solid triangle) in the cells treated with HCQ (100 µM) plus ant-Fas IgM (CH11, 100 ng/ml), which were determined by Western blotting analysis and DNA fragmentation enzyme-linked immunosorbent assay (ELISA), respectively. Data are presented as the mean of two independent experiments.