Fig. 2: Gene expression analysis of TREX1, cGAS and inflammatory cytokines in DNA-stimulated RA-FLSs transfected with or without TREX1 or cGAS siRNA.

A Time-dependent activation of the TREX1 and cGAS signalling pathways induced by DNA fragments. RA-FLSs were transfected with 5 μg of DNA fragments (sonicated DNA from RA-FLSs) for 24–72 h in the absence or presence of Lipofectamine 3000. Transfected RA-FLSs were then harvested for gene expression analysis of TREX1, cGAS and inflammatory cytokines using RT–PCR. All samples are biologically independent, and statistical significance was analyzed by one-way ANOVA, *P < 0.05, **P < 0.01 versus control groups. Data are presented as the mean ± s.e.m from three independent experiments. B Dose-dependent activation of the TREX1 and cGAS signaling pathways by DNA fragments. RA-FLSs were transfected with different amounts of DNA fragments (1 μg, 5 μg and 10 μg) in the presence of Lipofectamine 3000 for 24 h. Transfected RA-FLSs were then harvested for gene expression analysis of TREX1, cGAS and inflammatory cytokines using RT–PCR. All samples are biologically independent, and statistical significance was calculated by one-way ANOVA, *P < 0.05, **P < 0.01 versus control groups. Data are presented as the mean ± s.e.m. from three independent experiments. C Knockdown of TREX1 or cGAS reversed the time-dependent activation of the TREX1 and cGAS signalling pathways in DNA fragment-stimulated RA-FLSs. RA-FLSs were transfected with TREX1 siRNA or cGAS siRNA for knockdown of the corresponding gene 24 h prior to treatment with DNA fragments. Knockdown cells were then transfected with 5 μg of DNA fragments for 24–72 h. Transfected cells were then harvested and analysed by RT–PCR to determine the mRNA expression of inflammatory cytokines. All samples are biologically independent, and statistical significance was calculated by one-way ANOVA, *P < 0.05 for TREX1 knockdown cells compared with cells transfected with DNA fragments alone; #P < 0.05 for cGAS knockdown cells compared with cells transfected with DNA fragments alone. Data are presented as mean ± s.e.m. from three independent experiments. (T: TREX1 siRNA; C: cGAS siRNA).