Fig 2: SMAD3 K53/K333 methylation is essential for its membrane localization.(A) Membrane and cytosolic fractions from MDA-MB-231SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids were collected and subjected to IB analysis. (B) Membrane fractions from MDA-MB-231SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL) were collected and subjected to IB analysis. (C) MDA-MB-231SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). IF images show the cellular localization of SMAD3. Scale bar: 10 μm. (D) Membrane and cytosolic fractions from MDA-MB-231 cells silenced with control or EZH2 shRNA (no. 1) were collected and subjected to IB analysis. (E) HEK293T cells were transfected with WT HA-SMAD3 or HA-SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-HA antibody, followed by IB analysis. (F) HEK293T cells were silenced with control or EZH2 shRNA (nos. 1 and 2), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. (G) Co-IP of endogenous SMAD3 from MDA-MB-231 cells transfected with EZH2WT or EZH2H689A, followed by IB analysis. (H) Co-IP of endogenous SMAD3 from MDA-MB-231 cells transfected with EZH2WT or EHZ2Y641H, followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.

Fig 3: 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.

Fig 4: SMAD3 methylation levels are positively correlated with EZH2 and SMAD3 phosphorylation levels.(A) Scatter plot of the IHC staining scores for EZH2, SMAD3 K53/K333 trimethylation, and SMAD3S423/S425 phosphorylation in breast cancer (n = 75). All P and r values were calculated with Spearman’s r test. (B–D) Quantitative IHC staining scores showing the correlation between EZH2, SMAD3 K53/K333 trimethylation, and SMAD3S423/S425 phosphorylation. neg, negat5ive; pos, positive. (E) Quantitative IHC staining score for staining of SMAD3 K53/K333 trimethylation in nonmetastatic primary breast tumors and lung-metastatic primary breast tumors. P = 0.0199. Statistical significance was calculated with the χ2 test.

Fig 5: 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).