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. 2009 Jul 1;20(13):3115–3124. doi: 10.1091/mbc.E09-01-0046

Figure 2.

Figure 2.

Zyxin is essential for cell migration and actin fiber reorganization in TGF-β1–induced EMT. (A) Immunoblot analysis of zyxin and E-and N-cadherin in NMuMG-C7 cells transfected with zyxin siRNA or scramble RNA. After treatment with TGF-β1 for 24 h, total lysates were subjected to Western blot. GAPDH was used as a loading control. (B) Zyxin depletion abolished cell motility in TGF-β1-EMT. Cell motility was assessed by modified Boyden's chamber assay. NMuMG-C7 cells were transfected with zyxin siRNA or scramble RNA and treated or not with TGF-β1 for 24 h. The cells that migrated to the lower surface of the membrane were stained after 4 h of incubation. Scale bar, 250 μm. (C) The number of migrated cells in B. *p = 0.0344 versus Scramble RNA, TGF-β1 (−); **p = 0.0159 versus Scramble RNA, TGF-β1 (+). Results are shown as the mean ± SEM of four separate migration assays from two independent experiments. (D–K) Zyxin is essential for actin fiber formation in TGF-β1-EMT. Phalloidin staining was performed with NMuMG-C7 cells transfected with zyxin siRNA or scramble RNA and treated or not with TGF-β1 for 24 h. Apical and basal actin network were observed separately with a confocal microscope. Note that both the apical and basal actin fiber formation were hampered by zyxin depletion. Scale bar, 50 μm. (L) The scheme of EGFP-tagged zyxin deletion constructs. EGFP–zyxin constructs were introduced into NMuMG-C7 cells by transient transfection. FL: full length. (M) NMuMG-C7 cells were transduced with lentivirus vector that expresses shZyxin that targets 3′UTR. The resultant cells were then transfected with the EGFP–zyxin constructs that lack 3′UTR. The suppression of endogenous zyxin and the expression of transfected genes were confirmed by blotting with anti-zyxin and ant-EGFP antibodies. LIM only construct lacks the epitope recognized by zyxin antibody. (N) Neither ΔLIM nor LIM only constructs rescued the endogenous zyxin depletion. Endogenous zyxin-depleted cells were transfected with EGFP–zyxin constructs and treated with TGF-β1 for 24 h. Actin fiber formation was observed in the cells transfected with FL (arrowhead), but not with ΔLIM or LIM only constructs. Scale bar, 50 μm.