Figure 2. Reactivation of SIX1 and SIX2 by NIK results in inflammatory gene suppression.

a, qRT-PCR of total RNA isolated from WT or Nik^−/− primary BMDMs infected with Lm (MOI=0.1) or treated with 25 ng/ml TNF for 24 hours. The relative gene expression was normalized to untreated control. Bars are the mean from 2 independent experiments and circles are the average of 2 technical replicates from each experiment. b, c, Levels of Six1 protein in WT or Nik^−/− primary BMDMs infected with Lm (MOI=~0.1) for indicated time points (b) or for 24 hours (c). Whole cell lysate or nuclear extracts were probed with indicated antibodies by western blot. Quantification of Six1 protein levels (mean±s.d. from 3 independent experiments) in the indicated samples were normalized to WT cells (1.0). d, e, SIX1 expression levels in WT and NIK^−/− fibroblasts treated with 50 ng/ml TWEAK (d) or 5 μM BV6 (e) for 24 hours and processed for western blot analysis as in b. f, qRT-PCR of total RNA isolated from WT and SIX1^−/− SIX2^−/− fibroblasts treated with 50 ng/ml TWEAK for 24 hours. The relative gene expression was normalized to WT untreated control and shown as mean±s.d. of 3 technical replicates from one experiment. Data are representative of 3 independent experiments. g, qRT-PCR of total RNA isolated from WT and SIX1^−/− SIX2^−/− fibroblasts transduced with Fluc or NIK lentivirus for 72 hours. The relative gene expression was normalized to WT Fluc transduced control and shown as mean±s.d. of 3 technical replicates from one experiment. Data are representative of 3 independent experiments. All western blot data are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.