Validation of hairpin construct targeting tumor necrosis factor receptor 1 (HpTNFR1) in vitro. (a) NIH-3T3-5 × NF-κB-luciferase cells were transduced at indicated multiplicity of infection (MOI) HpTNFR1 or hairpin non specific (HpNS) and, after 2 days, stimulated with 10 ng/mL mTNFα for 6 hours. Nuclear factor-kappa-B (NF-κB)-driven luciferase activity is represented as mean ± standard error of the mean (SEM) (n = 4) of percentages compared with the HpNS group. The numbers of HpNS transduced cells (doses MOI 10) with or without TNFα stimulation were 164,232 ± 864 and 21,555 ± 864 relative light units (RLU)/mg protein, respectively. (b) Expression of TNFR1 in NIH-3T3-5 × NF-κB-luciferase cells transduced at indicated MOI with HpTNFR1. Data are represented as the mean (n = 10) of the difference in TNFR1 ΔCt values compared with HpNS-treated group (ΔΔCt). (c) NIH-3T3-5 × NF-κB-luciferase cells were transduced at MOI 10 with HpTNFR1 or HpNS or left untreated. After 2 days, untreated cells were preincubated for 1 hour with 10 μg/mL Enbrel, and thereafter all groups were stimulated with 10 ng/mL mTNFα or mIL-β for 6 hours. Luciferase activity is represented as mean ± SEM (n = 4). Statistical differences were determined using analysis of variance with Bonferroni post-test. *P < 0.05; ***P < 0.001. Ct, cycle threshold; IL, interleukin; TNF, tumor necrosis factor.