Fig 2: SMYD3 blockade with the BCI121 inhibitor hampers TGFß-induced EMT. (A) Epithelial (E-Cadherin, Claudin-6) and mesenchymal (N-Cadherin and Fibronectin) transcripts were quantified by quantitative real-time PCR analysis, relative to GAPDH. NMuMG cells were treated with 10 ng/ml TGFß in the presence of 0, 1, 5 10 and 50 µM BCI121, for 48 h. Statistical analysis was performed with one-way ANOVA, followed by post-hoc Tukey test. Data represent means±SD. n\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$ \ge$\end{document}3, * P = 0.05, ** P = 0.01, *** P = 0.001. (B) N-Cadherin and MMP9 transcript levels were quantified by quantitative real-time PCR normalized to GAPDH in MCF10A cells treated with 5 ng/ml TGFß in the presence of 0, 1, 5 10 and 50 µM BCI121 for 48 h. Data represent means±SD. Statistical analysis was performed with one-way ANOVA, followed by post-hoc Tukey test. n\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$ \ge$\end{document}3, * P = 0.05, ** P = 0.01, ***P = 0.001. (C, D) Wound healing assay on NMuMG and MCF10A cells treated with 10µM BCI121 and 5 and 10 ng/ml TGFß respectively, for 0 and 16 h. Migration percentage is reported in the right panel. Statistical significance was calculated with paired Student's t-test. * P = 0.05, *** P = 0.001. Scale bar = 10 µm.

Fig 3: Smyd3 overexpression induces PARP16-mediating ER stress. (A) RAECs were transfected with lentivirus-mediated Smyd3 cDNA (Smyd3 OE) for 72 h, cell lysates were immunoblotted with antibody against Smyd3, PARP16, p21, VCAM-1, p-PERK, Spliced XBP-1. All data were shown as mean ± S.D of at least 4 independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control. (B) Knockdown of PARP16 blocked ER stress and the upregulation of p21 upon overexpression of Smyd3. Smyd3 overexpressed RAECs were transfected with control or Smyd3 siRNA, and then induced by Ang II, cell extracts were collected for determining the protein levels of PARP16, p-PERK, Spliced XBP-1, and p21 by Western blot. Data were shown as mean ± S.D of at least 4 independent experiments. #p < 0.05, ###p < 0.001 vs. control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Smyd3 OE cells. (C) Inhibition of Smyd3 or PARP16 decreased RAECs senescence markers. Pretreated with PARP16 inhibitor (EGCG) or Smyd3 inhibitor (EPZ031686) for 4 h, RAECs were treated with Ang II for 48 h, respectively, cell extracts were collected for determining the protein levels of p21, p53, and VCAM-1 by Western blot. All data were shown as mean ± S.D of at least 4 independent experiments. ###p < 0.001 vs. control; *p < 0.05, ***p < 0.001 vs. Ang II treated cells.

Fig 4: SMYD3 is required for DNA repair machinery. (a) Whole-genome microarray analysis of RNAs isolated from shLuc vs. shSMYD3 MCF7 cells was conducted. With a cut-off of absolute normalized fold change \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\leqq $$\end{document}? 0.5 (log2 normalized ratios < -1), the list of down-regulated genes was further categorized by the DAVID v6.8 Gene Ontology program to reveal their gene ontology process. (b) ?H2A.X foci formation at indicated times after 1.67 Gy IR treatment in shLuc or shSMYD3 MCF7 cells. *P < 0.05. **P < 0.01. (c) ?H2A.X foci formation at indicated times after 1.67 Gy IR treatment in shLuc or shSMYD3 MCF7 cells complemented with the vector control, SMYD3 or mutant SMYD3Y239F. *P < 0.05. **P < 0.01. ***P < 0.001. (d–f) Clonogenic survivals of shLuc and shSMYD3 MCF7 (d), MDA-MB-231 (e) and AU565 cells (f) treated with indicated dosages of IR. All values in the diagrams were means ± SD of triplicates and data were representative of n = 3 for each experiment.

Fig 5: SMYD3 knockdown downregulates HR gene expressions. (a) The confirmation of the microarray analyses for the expressions of candidate genes in the shSMYD3/shLuc dataset using qRT-PCR. The fold changes of each gene expression in the microarray data were listed below. *P < 0.05, **P < 0.01, ***P < 0.001 vs. shLuc control. (b) Western blot analyses of SMYD3, MDC1, EXO1 and RAD54B in shLuc and shSMYD3 MCF7 cells. Tubulin was used as a loading control. The ratio of individual protein relative to the loading control tubulin was indicated below. (c,d) DNA repair foci formation of MDC1 foci (c) and BRCA1 foci (d) formation at indicated times after 1.67 Gy IR treatment in shLuc or shSMYD3 MCF7 cells. *P < 0.05. **P < 0.01. ***P < 0.001. All values in the histograms were means ± SD of triplicates and data were representative of n = 3 for each experiment.