Figure 5.
FOSL1 deficiency dampens mutant Kras–induced AREG expression in lung tumors, and FOSL1 promotes KRAS mutant HLAC cell proliferation by regulating AREG expression. (A) Areg mRNA expression in lung tumors of KrasG12D mice and Fosl1F/F:KrasG12D mice at 13 weeks and 20 weeks after Kras oncogene activation. Values are expressed relative to the lung tissue of KrasG12D mice (notated as Cont) without adenoviral Cre infection. Data are mean ± SD (n = 3–6). *P ≤ 0.05 versus Cont; †P ≤ 0.05 versus Kras. (B) AREG expression in H2009 cells transfected with si-SCR or si-FOSL1 for 72 hours. Data are mean ± SD (n = 3–4). *P ≤ 0.05 versus si-SCR. (C) H2009 cells (15,000/well) transfected with SCR siRNA or AREG siRNA (100 nM) for 24 hours were seeded onto a 96-well plate. Cells were harvested at 24 hours and 48 hours thereafter, and their proliferation was measured by CellTiter-Glo assay (left) or cell counting (right). Data are mean ± SD. *P ≤ 0.05, versus si-AREG. (D) To measure clonogenicity, H2009 cells (1,500/well) transfected with si-SCR or si-AREG for 24 hours were seeded onto a 12-well plate and cultured for 7 days. Colonies were stained with crystal violet, dried, and photographed (left), and the number of colonies was enumerated (right). Data are mean ± SD. *P ≤ 0.05 versus si-SCR. (E) Exogenous AREG improves HLAC cell survival imparted by FOSL1 deficiency. H2009 cells transfected with FOSL1 siRNA were seeded on a 96-well or 12-well plate and then cultured in the presence of recombinant AREG (rAREG; 50 ng/ml) or vehicle (PBS) for 3 days to measure their proliferation rate (left) or 7 days to measure clonogenicity (right) as in Figure 4. Data are mean ± SD. *P ≤ 0.05, versus PBS. **P = 0.06 versus PBS.