Figure 4. The E2A proteins and TWIST1 reciprocally stabilize each other.

(A) Silencing of TCF3 leads to downregulation of TWIST1 in the KRAS mutant NSCLC cell line, A549. Cells were infected with the indicated shRNAs and were harvested for western blot analysis 96 hours following infection. (B) Overexpression of the E2A proteins, E12 or E47 induces upregulation of TWIST1 in 293T cells as well as in KRAS mutant NSCLC cells (A549, H460). 293T cells were harvested 72 hours following transfection. Experiments in A549 and H460 were performed in stable E12 or E47 overexpressing cell lines. (C) Silencing of TWIST1 induces downregulation of E2A proteins in KRAS mutant (A549, H460) as well as in MET amplified (H1648, H1993) NSCLC cell lines. Cells were infected with the indicated shRNAs and were harvested for western blot analysis 96 hours following infection. (D) Overexpression of TWIST1 induces the E2A proteins in KRAS mutant NSCLC cells (H460) and EGFR mutant NSCLC cells (PC-9). H460 cells were harvested once stable cell lines were established. PC9 TRE3G-TWIST1 cells were harvested following 500 ng/ml treatment of doxycycline for 24 hours. (E) LEFT: TWIST1 induction of E2A proteins is not accompanied by a robust upregulation of TCF3 mRNA in KRAS mutant (H460) and EGFR mutant NSCLC cells (PC-9). All TWIST1 overexpressing groups are normalized to untreated group (n=3 technical replicates). Data represent mean ±SD. RIGHT: TWIST1 overexpression increases the half-life of E2A proteins. Protein concentration was quantified using densitometry and protein half-lives were determined using linear regression.