Figure 4.

(A and B) Suppression of Klf4 reduces survival of cultured MLE-12 cells. (A) Transfection with siRNA against Klf4 using lipofectamine reduces Klf4 protein abundance in MLE-12 cells 72 hours after transfection compared with cells transfected with noncoding siRNA (control). Klf4 protein was assessed by immunoblot and related to β-actin (mean and SD; n = 3; *P < 0.05). (B) The cell content of p21 protein, a downregulator of cell-cycle progression, is increased in Klf4-depleted cells compared with Klf4-replete cells after 72 hours in serum-rich culture medium (three blots per group, shown from a total of n = 6 experiments; mean and SD; *P < 0.05). (C and D) Klf4 suppression by siRNA transfection results in decreased proliferation and increased apoptosis of MLE-12 cells cultured for 24 hours in serum-reduced medium compared with Klf4-expressing (control) cells. Cell proliferation was measured by the MTT assay (n = 5; mean and SD; *P < 0.05). MLE-12 cell apoptosis, measured by the Caspase-Glo 3/7 assay, is increased in Klf4-depleted cells compared with Klf4-replete cells after 72 hours in culture with serum-rich medium (n = 6; mean and SD; *P < 0.05). (E) Inhibition of EGFR phosphorylation reduces Klf4 protein content in cultured mouse lung epithelial cells. Cells were grown to confluence and exposed to Tyrphostin (AG1478, 10 μM) for 24 hours in serum-rich medium. Klf4 protein was assessed by immunoblot, and quantitative densitometry was performed for two bands, one at 52 kD (left panel) and another at 65 kD (right panel), relative to β-actin. Two representative immunoblots/group, and the corresponding densitometric measurements for each band obtained from five studies are displayed (n = 5; mean and SD; *P < 0.05).