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. 2022 Mar 1;132(5):e152394. doi: 10.1172/JCI152394

Figure 4. EZH2 promotes cell migration and invasion dependent on methylation of SMAD3 at K53 and K333.

Figure 4

(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. (FH). 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).