Fig 2: Deletion of SMAD3 K53/K333 methylation inhibits the SMAD3 oncogene in vitro and in vivo.(A) MDA-MB-231SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). Quantitative RT-PCR analysis of TGFB/SMAD3 signaling pathway downstream genes, including CTGF, PAI1, PDGFB, and SMAD7, in the indicated cells with or without TGFB1 (5 ng/mL) treatment. (B) Quantitative analysis of Transwell assay in the indicated cells. (C) IF and (D) IB analysis of EMT markers in the indicated cells. Scale bar: 50 um. (E and F) Representative lung image (E) and H&E-stained lung sections (F). Scale bars: 5 mm. (G and H) Scatter plots showing lung metastatic nodules (G) and lung weights (H). All immunoblots were performed 3 times, independently, with similar results. Data indicate the mean ± SD. **P < 0.05, by 2-tailed Student’s t test (A, B, G, and H).

Fig 3: EZH2 promotes cell migration and invasion dependent on methylation of SMAD3 at K53 and K333.(A) Quantitative analysis of Transwell assay in the indicated MDA-MB-231 cells treated with TGFB1 (5 ng/mL). (B) MDA-MB-231SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and silenced with control or EZH2 shRNA (nos. 1 and 2). WCEs were collected for IB analysis. (C) WT Flag-EZH2 or a Flag-EZH2 H689A plasmid was transfected into MDA-MB-231SMAD3–/– cells ectopically expressing WT SMAD3 or SMAD3 K53/333R, and WCEs were collected for IB analysis. (D and E) A Transwell cell invasion assay was performed using MDA-MB-231SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and transfected with a vector, EZH2WT, or EZH2H689A. Representative images (D) and quantitative analysis (E). Original magnification, ×200. (F–H). Representative lung image (F),H&E-stained lung sections (G), and scatter plot showing lung weights (H). Scale bars: 5 mm. All immunoblotting was performed 3 times, independently, with similar results. Data indicate the mean ± SD. **P < 0.05, by 2-tailed Student’s t test (A, E, and H).

Fig 4: SMAD3 K53/K333 methylation predicts poor survival of patients with cancer.(A) Kaplan-Meier survival analysis comparing high and low levels of SMAD3 K53/K333 trimethylation using microarray results from a breast cancer specimen. P = 0.0004. (B) Cox regression analysis showing the significance of the relationship between SMAD3 K53/K333 trimethylation expression and prognosis for patients with breast cancer in the presence of other clinical variables. (C) Scatter plot of SMAD3 K53/K333 trimethylation IHC staining score comparing different N stages using microarray results from a breast cancer specimen (n = 80). P = 0.0051, by χ2 test. (D) Scatter plot of the number of cancer-positive lymph nodes comparing high and low levels of SMAD3 K53/K333 trimethylation using microarray results from a breast cancer specimen (n = 80). P = 0.0039, by χ2 test. (E) Working model of EZH2-mediated SMAD3 K53/K333 methylation crosstalk with TGFB-mediated SMAD3 phosphorylation, membrane localization, and activation.

Fig 5: SMAD3 methylation is triggered by EZH2.(A) WCEs of MDA-MB-231 and MCF-7 cells were collected and subjected to co-IP and IB assays. (B) HEK293T and MCF-7 cells were serum starved and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (C) MDA-MB-231 and MCF-7 cells were treated with TGFB1 (5 ng/mL) and the EZH2 inhibitors GSK126 or GSK503, and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (D) HEK293T cells were transfected with WT HA-SMAD3 or mutant plasmids and a Flag-EZH2 plasmid as indicated/WCEs were then collected for IP with anti-HA antibody, followed by IB analysis. (E) Immunoprecipitated EZH2 from HEK293 cells was incubated with SAM along with SMAD3 protein for in vitro methylation of SMAD3. The methylated proteins were separated by SDS-PAGE, and SMAD3 methylation was analyzed by IB using anti–SMAD3 K53/K333 trimethylation–specific antibodies. (F) MDA-MB-231 cells silenced with control (shNC) or EZH2 shRNA (nos. 1 and 2) were treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (G) HEK293T cells were transfected with vector, EZH2WT, or EZH2H689A and then treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (H) HEK293T cells were transfected with vector, EZH2WT, or EZH2Y641H, and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.