Figure 2. SIRT6 Directly Interacts with PPARα and Binds to and Activates the PPRE In Vivo.

(A) In vitro pulldown assay of SIRT6-FLAG and co-immunoprecipitation of GST-PPARα from recombinant proteins.

(B) Microfluidics in vitro association assay. SIRT6-FLAG was fixed onto the chip, and Myc-tagged associated proteins were incubated and then washed. Interaction ratio was detected by fluorescence (left). Representative fluorescence binding on chip (right).

(C) Co-immunoprecipitation of FLAG-tagged SIRT6 and GFP-tagged PPARα.

(D) Co-immunoprecipitation of FLAG-tagged PPARα and endogenous SIRT6 from HEK293T cells.

(E) Microfluidics assay of SIRT6 binding to PPRE or mutant sequence in the presence/absence of PPARα and representative fluorescence binding on chip.

(F) Luciferase activity of PPRE promoter in HEK293T cells overexpressing either SIRT6 WT or dominant-negative (DN) mutant.

(G) Luciferase activity in HEK293T cells overexpressing SIRT6 and increasing amounts of PPARα.

(H) Srebp2 and Gapdh were used as positive/negative controls, respectively.

(I) ChIP-quantitative real-time PCR analysis of H3K9 acetylation on PPREs of indicated genes in WT and Sirt6^+/− livers.

In (B), data are represented as means + SE and significance was calculated by one-way ANOVA followed by a Bonferroni multiple comparisons test; n = 10. In (E)–(I), data are represented as means + SE. In (E) and (I), two-way ANOVA followed by a Bonferroni multiple comparisons test were used; n = 10 for (A) and (E) and n = 3 for (I). In (H), significance was calculated by two-tailed Student’s t test. *p < 0.05, **p < 0.01.