Susceptibility of proliferating rheumatoid arthritis synovial fibroblasts (RASFs) to Fas ligand (FasL)-induced and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. (a) As assessed by Cell Death Detection (ELISAPlus), higher-proliferating RASFs (50% of confluency) were less sensitive to TRAIL-induced apoptosis than lower-proliferating RASFs (80% of confluency) and significantly less sensitive than confluent RASFs (100% confluent). (b) As revealed by the Apo-ONE® Homogeneous Caspase-3/7 Assay, higher-proliferating RASFs showed lower activities of caspase-3 and caspase-7 after induction of apoptosis with FasL than less-proliferating RASFs and confluent RASFs. Highly synchronised RASFs in S phase (HU/24 h) or G2/M phase (HU/30 h) were less sensitive to TRAIL-induced (c) and FasL-induced (d) apoptosis than synchronised RASFs in G0/G1 phase (HU/0 h), as measured by the Apo-ONE® Homogeneous Caspase-3/7 Assay. Moreover, RASFs arrested in G0/G1 phase through serum deprivation using insulin-transferrin-sodium selenite (ITS) medium (8 d) were more sensitive to TRAIL-induced (e) and FasL-induced (f) apoptosis than proliferating RASFs in S phase (9/1 d) or G2/M phase (9/2 d). Staurosporin-induced apoptosis was measured as a positive control. All values are mean ± standard deviation of fluorescence/fluorescence of unstimulated RASFs from at least three independent patients with rheumatoid arthritis. *P < 0.05, **P < 0.01, ***P < 0.001.