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

Figure 3.

Figure 3

K20/K117 acetylation of SMAD3 upregulates immune response‐related cytokines. A) Heatmap of mRNA‐Seq analysis of differentially expressed genes regulated by SMAD3 K20/117Q (SMAD3‐2KQ) (twofold change and FDR < 0.05). SMAD3 sgRNA‐resistant Flag‐SMAD3 WT and K20/117Q (2KQ) mutant were infected into sgSMAD3/MDA‐MB‐231 cells without TGF‐β1 treatment. B) Gene ontology (GO) analysis indicates that the genes upregulated by the SMAD3‐2KQ mutant were associated with inflammatory and immune response‐related signaling pathways. C) Hierarchical clustering analysis of patient gene expression data (TCGA) indicates that the 14‐gene signature resulted in two main clusters indicated in blue and red, respectively. Expression data of 17 TNBC patients with metastasis (+M) and recurrence (+R) and 144 TNBC patients without metastasis (−M) and recurrence (−R) were downloaded from TCGA. D) GSEA analyses showing that KAT6A‐acetylated SMAD3 upregulates IL‐6, IL‐22, and TNFα signaling pathways. NES, normalized enrichment score. E) qRT‐PCR analyses of upregulation of IL‐6, IL‐22, and TNFA by SMAD3‐2KQ mutant in MDA‐MB‐231/sgSMAD3 and BT‐549/sgSMAD3 cells without TGF‐β1 treatment. F) Enzyme linked immunosorbent assay (ELISA) of expression of IL‐6, IL‐22, and TNFα in the condition medium from cells in panel (E). G) In panels (E) and (F), data are representative of three independent experiments with similar results. Error bars, SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by paired two‐tail t‐test.