<i>TWIST1</i> knockdown activates senescence in human non-small cell lung cancer cells.

<p>Three different shRNAs were able to knockdown <i>TWIST1</i> mRNA levels and result in decreased TWIST1 protein in the <i>KRAS</i> mutated non-small cell lung cancer (NSCLC) cell line H460 as shown by (A) qPCR and (B) TWIST1 Western blotting on day 9 after the shRNA infection. (C) Representative duplicates of crystal violet staining of serially diluted H460 NSCLC cells demonstrate <i>TWIST1</i> knockdown decreases cellular proliferation. (D) Representative photomicrographs of increased SA-β-gal staining of cells following shRNA mediated <i>TWIST1</i> knockdown using sh-TWIST1-39. (E) Quantification of SA-β-gal stained cells following shRNA mediated <i>TWIST1</i> knockdown. (F) <i>TWIST1</i> knockdown in H460 results in the upregulation of some additional markers of senescence, p21 and p27 as shown by Western blotting on day 9 after the shRNA infection. (G) A549 cells require <i>TWIST1</i> overexpression to form subcutaneous tumors in NOD-SCID mice. A contingency table of A549 cells infected with sh-Scrambled control or sh-TWIST1 shRNA that were implanted into NOD-SCID mice and 4 weeks later scored for tumor development (5/6 <i>versus</i> 0/5, respectively, <i>p</i> = 0.01 by Fisher's exact test).