Extended Data Fig. 7: Doxycycline-induced HA–SIX1 expression in mice.
From: A NIK–SIX signalling axis controls inflammation by targeted silencing of non-canonical NF-κB
a, Doxycycline-induced HA–SIX1 bitransgenic mouse model system. CAG–rtTA3 mice were intercrossed with Tet-on driven HA–SIX1 mice to obtain rtTA3+/−SIX1+ mice. In principle, doxycycline-bound rtTA3 targets the Tet-on operator and drives broad HA–SIX1 expression across multiple tissues. Primer sets used to genotype CAG–rtTA3 on chromosome 8 and Tet-on HA–SIX1 are shown. Electrophoresis gels show genotyping of a representative rtTA3+/−SIX1+ mouse. b, Anti-SIX1 western blot of whole cell lysates from BMDMs isolated from rtTA3+/−SIX1+ mice. HA–SIX1 is not expressed in the absence of doxycycline under quiescent condition (left). When TNF was administered to these cells, endogenous SIX1 was stimulated (right). c, Whole cell lysates from doxycycline-treated BMDMs isolated from rtTA3+/− or rtTA3+/−SIX1+ mice. BMDMs were stimulated with TNF as indicated and probed with anti-SIX1 antibody by western blot. Doxycycline induced HA–SIX1 expression (lane 1), and this induction was potentiated by TNF (lane 2). HA–SIX1 ran as a doublet, which potentially represents an unmodified and a mono-ubiquitinated form of SIX1. Neither endogenous SIX1 nor HA–SIX1 was detected in BMDMs isolated from Dox-treated rtTA3+/− mice (lane 3). In control experiments, TNF induced endogenous SIX1 in BMDMs isolated from Dox-treated rtTA3+/− mice (lane 4). d, rtTA3+/−SIX1+ and rtTA3+/− mice were given 2 mg ml−1 doxycycline in drinking water for 10 days. Cell lysates from liver and spleen were used to probe HA–SIX1 expression by anti-SIX1 western blot. All western blot data are representative of three independent experiments. e, Peritoneal macrophages were isolated from rtTA3+/−SIX1+ and rtTA3+/− littermate control mice. Adherent macrophages were incubated with 2 μg ml−1 doxycycline for 24 h and then mock-treated or treated with 100 ng ml−1 LPS for 4 h. Total RNA was isolated for qRT–PCR. Relative gene expression was normalized to rtTA3+/− untreated control. Bars show mean from two technical replicates (shown as circles). Data are representative of three independent experiments. f, Experimental procedures corresponding to Fig. 4a–c (see Methods). For gel source data, see Supplementary Fig. 1.
