For clinical relevance, we evaluated FLS derived from synovium explants of three RA patients, for their sensitivity to PUMA-induced cell death. BVCARHAdV5-PUMA transduction was carried out at a constant vector dose of 50 vp/cell, with or without pretreatment with proinflammatory cytokines, and the cell survival measured by the MTT assay over a period of 40 h post infection. The cell survival curves from the three clinical samples show similar profiles, with a rapid cell death at 16 h post infection, followed by a more gradual effect until 40 h (Fig. 3a–c). The percentage of surviving cells at 40 h post transduction was 20% or less in all three samples, and minor differences in the cell survival curves observed in the presence or absence of cytokines (Fig. 3a–c). The results show that BVCARHAdV5-PUMA transduction of the FLS derived from the three clinical samples induced rapid cell death in all samples, with or without cytokines.Fig. 3BVCARHAdV5-PUMA-induced apoptosis of FLS derived from clinical samples. FLS derived from synovium explants from three patients with RA a–c, nontreated or treated with cytokines TNFα and IL-17, were infected with BVCARHAdV5-PUMA at a constant adenoviral vector dose of 50 vp/cell. The cell survival was monitored over 40 h, using the MTT assay. The number of surviving cells was expressed as the percentage of control cells taken at time 0 of infection, which was attributed the 100 per cent value. The average from three independent experiments, each performed in triplicates is presented. The error bars indicate standard error of the mean. Statistical analysis using one-way ANOVA test, ***P < 0.001, **P < 0.01, *P < 0.05. d The expression of PUMA protein was analysed in FLS transduced or not with BVCARHAdV5-PUMA at 50 vp/cell. Cells were harvested at 40 h post transduction, lysed and whole-cell lysates probed for PUMA protein using SDS–polyacrylamide gel electrophoresis and western blot analysis. (bottom) Luminogram of blot reacted with anti-PUMA RabMAb, peroxidase-conjugated anti-rabbit IgG antibody and enhanced chemiluminescence Detection Kit. (Top) Loading control (stained blot). Blot was reacted with anti-β-actin MoMAb, peroxidase-conjugated anti-mouse IgG antibody, and staining reaction performed with H202 and 3,3’-Diaminobenzidine. e To quantitatively measure cell apoptosis, FLS untreated or treated with individual cytokines TNFα and IL-17, or a mixture of TNFα and IL-17, were incubated with increasing doses of BVCARHAdV5-PUMA, and harvested at 24 h post infection. DNA fragmentation and nucleosome release into the cytoplasm were determined using an immunological detection of histone-complexed DNA fragments. Lysates from mock-infected cells served as negative controls to evaluate the background level, i.e., the physiological nucleosome content of the cytoplasm. The results are expressed as the fold change over the background level, which was attributed the value of 1. The control consisted of FLS infected by BVCARHAdV5-GFP at the highest dose of 50 vp/cell. Results presented are from three independent experiments, each in triplicates, using the one-way ANOVA test, ***P < 0.001, **P < 0.01, *P < 0.05