Fig 1: p300‐mediated acetylation is vital for Smad1 oncofunctions. A) IP assay detecting the acetylation of Smad1 in indicated GBM cell lines. B) IP assay indicating Smad1 acetylation is regulated by p300. Indicated cells were transfected with p300 siRNA, followed by IP using anti‐Smad1 antibody and IB using the indicated antibodies. The relative quantification of Smad1 acetylation was listed. C) Schematic diagram of p300‐specific acetylation site prediction of Smad1 and its mutants with unit point or multi‐site mutation of the indicated Lysine (K) residues converted to Arginine (R). D) IP assay detecting the acetylation of Smad1 mutants. U87 cells were transfected with indicated Smad1 constructs, followed by IP using anti‐Flag antibody and IB using the indicated antibodies. The relative Smad1 acetylation was listed under the bands. E) SBE reporter assay monitoring the activity of SMAD responsive transcriptional regulation in U87 cells expressing the indicated constructs (n = 3, ** p < 0.01). F) Western blot analysis of ID1 expression in U87 cells expressing the indicated constructs (n = 3, ** p < 0.01). G) ChIP‐qPCR assay detecting the enrichment of p53 binding to the promoter of ID1 in U87 expressing different Smad1 constructs (n = 3, ** p < 0.01). H) Cell growth assay of p53 KO cells overexpressing indicated Smad1 constructs. Cells were cultured for 48 h, and followed with a CCK‐8 assay to detect cell number (n = 6, ** p < 0.01). I) Representative colony formation (left panel) of p53 KO cells overexpressing indicated Smad1 constructs (n = 3, ** p < 0.01). J) Apoptosis assay of indicated GBM cells. Cells were treated with Dox (2 µM) for 24 h and apoptotic cells were analyzed by FACS (n = 3, ** p < 0.01). K) Western blot analysis detecting p53 acetylation in indicated GBM cells expressing different Smad1 constructs. The relative quantifications of p53 acetylation were listed. L) Diagram illustrating the relationship among p53, Smad1 and p300 in regulating acetylation of p53 and Smad1. Smad1 binds p53 and p300 via MH1 and MH2 domain, respectively, and destroys the interaction of p53 and p300. MH2 is essential, but MH1 is dispensable for Smad1 mediated p53 acetylation inhibition.