Figure 4. Overexpression of Snai1 in PC-3/Mc cells induces EMT and suppresses anchorage-independent growth and the expression of a self-renewal gene program.
(A) Overexpression of Snai1, Twist1, or TWIST2 in PC-3/Mc cells induced a fibroblastoid morphology and a downregulation of membrane-associated E-cadherin. Cells were transduced with retroviruses for the expression of mouse Snai1 or Twist1 or human TWIST2. Controls were PC-3/Mc cells transduced with pBABE and selected for puromycin resistance. Scale bars: 20 μm. (B) Overexpression of Snai1 strongly induced the invasiveness of PC-3/Mc cells, with a moderate effect by Twist1 or TWIST2. (C) Overexpression of Snai1 strongly inhibited spheroid growth by PC-3/Mc cells, with a moderate effect by TWIST2. (D) Overexpression of Snai1 in PC-3/Mc cells caused a strong downregulation of cell-surface E-cadherin, with a moderate effect by Twist1 or TWIST2, as determined by flow cytometry. (E) Overexpression of Snai1 in PC-3/Mc cells induced a downregulation of E-cadherin and EpCAM, a modest downregulation of SOX2 and MYC, and an upregulation of fibronectin and SPARC, as determined by Western blotting. Overexpression of Twist1 or TWIST2 induced a moderate downregulation of E-cadherin. (F) Overexpression of Snai1 and, more moderately, Twist1 or TWIST2, caused a downregulation of self-renewal and epithelial genes and an upregulation of mesenchymal genes. Relative transcript levels are represented as the log10 of ratios between experimental and control cells of their 2–ΔΔCp real-time PCR values. The levels of SNAI1 correspond to the endogenous, human transcripts, downregulated by overexpression of the exogenous (mouse) Snai1. Asterisk in F indicates that values for ectopic TWIST2 are off scale. Results are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.