Skip to main content
. 2021 Aug 13;8(20):2100014. doi: 10.1002/advs.202100014

Figure 4.

Figure 4

The K20/117 acetylation increases SMAD3 association with KAT6A‐mediated H3K23ac reader TRIM24 and thereby enhances SMAD3 activity. A) Oncogenic TRIM24 overexpression enhanced its association with SMAD3 and disrupted tumor suppressor TRIM33–SMAD3 interaction in MDA‐MB‐231 and BT‐549 cells stimulated with TGF‐β1. B) GST pull‐down assay was performed using purified GST‐SMAD3 and (His)6‐TRIM24, followed by WB with anti‐TRIM24 and anti‐SMAD3 antibodies. C) IP and WB of TRIM24 binding with SMAD2/3/4 in HEK293T cells stimulated by TGF‐β1. D) IP and WB of the effect of the K20/117Q (2KQ) or K20/117R (2KR) mutant on SMAD3 binding with TRIM24 and TRIM33 in HEK293T cells. E) IP and WB of the effects of the 2KQ or 2KR mutant on SMAD3 binding with TRIM24 and TRIM33, and IL‐6, IL‐22, and TNFα expression in MDA‐MB‐231 and BT‐549 cells. F) Effects of re‐expression of shRNA‐resistant Flag‐TRIM24 WT, F979A/N980A mutant, or an empty vector control (EV) on TRIM24 association with H3K23ac and SMAD3 in MDA‐MB‐231/tetO‐shKAT6A cells. G) Ectopic expression of TRIM24 F979A/N980A mutant decreases H3K23ac association with TRIM24 and SMAD3 in MDA‐MB‐231 cells. H) ChIP‐qPCR analyses of effects of ectopic expression of TRIM24 F979A/N980A mutant on SMAD3 binding to the promoters of IL‐6, IL‐22, and TNFA in MDA‐MB‐231 cells. I) Ectopic expression of TRIM24 F979A/N980A mutant decreased SMAD3‐mediated expression of IL‐6, IL‐22, and TNFA. Data are representative of three independent experiments with similar results. Error bars, SD. *P < 0.05, **P < 0.01, and ***P < 0.001 by paired two‐tail t‐test.