Fig. 7
Suv39h1 and SirT6 regulate IkBα expression upon NF-κB pathway activation. a Quantitative RT-PCR analysis (n = 3, SD) of mRNA from 293F cells transfected with scramble shRNA or shSuv39h1 and treated with TNFα. Cells were harvested at the indicated times of TNFα treatment. A representative group of early genes activated by NF-κB pathway upon TNFα induction were analyzed. b Same experiment as in a of IκBα expression upon TNFα induction in WT and Suv39h KO MEFs (n = 3, s.d). c Western blotting analysis of the levels of IκBα,phospho-IκBα (P-IκBα), p100, p52 and H3K92m3 in WT and Suv39h KO MEFs treated with TNFα the indicated times. Tubulin and histone H3 were used as loading controls. d ChIP analysis of the indicated factors in the IκBα promoter of 293F cells in non-induced conditions (ctrol) or upon treatment of TNFα for 1 and 2 h. Given the lack of a reliable Suv39h1 antibody, in this case we transfected the cells with Myc-Suv39h1 and performed the ChIP analysis with α-myc antibody. The genomic IκBα promoter was quantified with qRT-PCR(representative of n = 3; T-test; SEM, **p < 0.01, ***p < 0.005, ****p < 0.001). e ChIP analysis of IκBα promoter of Suv39h1 in 293F cells expressing shRNA scramble, shSirT6, or shSKP2 and analyzed as in d (n = 5; T-test; SEM, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001). f ChIP analysis of IκBα promoter with the indicated antibodies of 293F cells transfected with Myc-Suv39h1 either WT or the 8C mutant described in Fig. 3 upon 1 h of TNFα induction(representative of n = 3; T-test; SEM, ****p < 0.001). g Rescue experiment of human 293F expressing shScramble or shSuv39h1 with mouse Suv39h1 WT or 8C. Relative expression levels of IkBα upon 1 h of TNFα induction relative to the expression levels in shScramble cells. ((n = 5; T-test; SEM, *p < 0.05; **p < 0.01; ****p < 0.001). h Model of the novel mechanism proposed for the regulation of IκBα expression by the interplay between Suv39h1, SirT6, and SKP2
