Fig. 6

c-Jun/SIRT6/BAF170 complex binds to injury-specific enhancers and activates shh transcription in SCs. a RT-qPCR analysis of shh mRNA level in SCs treated with c-Jun overexpression or SIRT6 knockdown (n = 3). b 3D modeling of the interaction between c-Jun (blue) and SIRT6 (yellow) proteins elucidated through protein-protein docking predictions from AlphaFold 2. The stick representation delineates the amino acid residues, while yellow dashed lines illustrate the hydrogen bonds. c Western blot showing co-immunoprecipitation between SIRT6 and c-Jun. d Immunofluorescence images of the colocalization of SIRT6 and c-Jun in SCs. Scale bar: 10 μm. e RT-qPCR analysis of shh mRNA level in SIRT6 knockdown SCs rescued with different alleles of SIRT6 (n = 3). f Western blot showing co-immunoprecipitation between SIRT6 and BAF170 with downregulation or upregulation of SIRT6. g RT-qPCR analysis of Shh mRNA expression after BAF170 knockdown (n = 3). h Western blot showing the expression of BAF170 and Shh after BAF170 knockdown. i RT-qPCR analysis of shh mRNA level in SCs treated with SIRT6 knockdown and BAF170 overexpression (n = 3). j Western blot showing the expression of Shh, Ptch1, and Gli1 in SCs treated with SIRT6 knockdown and BAF170 overexpression. k RT-qPCR analysis of shh mRNA level in SCs after transfection with a plasmid expressing BAF170-K312A (n = 3). l Western blot showing the expression of Shh, Ptch1, and Gli1 in SCs treated with BAF170-K312A mutant. m Western blot showing co-immunoprecipitation between c-Jun and BAF170. n ChIP-qPCR for c-JUN and SIRT6 on three reported injury-specific shh enhancer sites and ChIP-qPCR for BAF170 on shh enhancer sites treated with SIRT6 knockdown (n = 3). o Luciferase reporter assay for shh enhancer 1 and 3 (n = 3). p Schematic showing the mechanism that c-Jun/SIRT6/BAF170 complex binds to injury-specific enhancers and activates SCs shh transcription. Data were presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001