Fig. 4

SIRT7 de-acetylates SMAD4 to regulate IRE1α transcription. a Relative mRNA level of IRE1α in A2058 and A375 cells with indicated treatment. b Three SMAD4 binding sites were identified in IRE1α promoter through transcription factor analysis. c Co-immunoprecipitation analysis of complex formation between endogenous SMAD4 and His-tagged SIRT7 immunoprecipitated from A2058 cells with indicated treatment. d Immunofluorescence staining of SIRT7 and SMAD4 in A2058 cells. Scale bar, 50 μm. e Co-immunoprecipitation analysis of acetyl-SMAD4 in A2058 cells with or without the knockdown of SIRT7. f Immunoblotting analysis of SMAD4 and SIRT7 in WM35 cells with or without SIRT7 overexpression treated with MG132 (10 μM) for 6 h. g Immunoblotting analysis of SMAD4 in A2058 cells with or without the knockdown of SIRT7 pre-treated with CHX (50 μg/mL) for 0 h, 4 h and 8 h. h Co-immunoprecipitation analysis of interaction between SMAD4 and K48-linked ubiquitin chain in A2058 cells with or without the knockdown of SIRT7 and WM35 cells with or without SIRT7 overexpression with MG132 treatment. i Chromatin immunoprecipitation analysis of the enrichment of SMAD4 to the promoter of IRE1α in A2058 cells with indicated treatment. j Relative mRNA level of IRE1α in A2058 cells with or without the knockdown of SIRT7 or SMAD4 under the treatment with TM (3 μM) for 24 h. k Immunoblotting analysis of IRE1α in A2058 cells with or without the knockdown of SIRT7 or SMAD4 treated with TM (3 μM) for 24 h. l Immunohistochemical staining and correlation analysis of SIRT7 and SMAD4 in TMA. Scale bar, 100 μm. Data represent the mean ± SD of triplicates. r value was calculated by Spearman correlation. P value was calculated by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001