Fig. 6

SIRT6 affects OCT4 transcriptional activity on pluripotency genes. a SIRT6 binding to upregulated genes in Sirt6-null iPS-like cell line and Sirt6-null MEFs. SIRT6 ChIP-seq published datasets [20] were analyzed by combining with the transcriptome datasets in Fig. 5. b Venn diagrams showing the overlapped associated genes of SIRT6 and OCT4 in mouse ES cells [24]. c Coimmunoprecipitation results showed that SIRT6 bound with OCT4. FLAG-tagged OCT4 and MYC-tagged SIRT6 were co-transfected in 293T cells and immunoprecipitation was performed by MYC antibody, and Western blotting was performed by indicated antibodies. d A luciferase reporter experiment showed the upregulation of Oct4 transcriptional activity on a Nanog promoter reporter and inhibited by co expressed with Sirt6. Oct4 was co-transfected with a Nanog reporter into 293T cells with Sirt6 or with empty vector control; firefly luciferase activity represented Nanog reporter activation level. Data represented as mean ± SD from three independent assays. e Real-time PCR for mRNA of Zfp42 in Sirt6-null iPS-like cell line transfected with Zfp42 siRNAs (Zfp42 KD) or negative control (NC) siRNA. f Percentage of OCT4-GFP-positive cells after 2 days of RA-induced differentiation of Sirt6-null iPS-like cells transfected with Zfp42 siRNAs (Zfp42 KD) or negative control (NC) compared to wild-type iPSCs. Representative flow cytometry studies are shown on the left, and the results of three replicates are summarized in the graph (*, p < 0.05; unpaired t test)