Fig. 5

Inhibition of EGFR signaling increases apoptosis in Foxf2-depleted cells. a Depletion of Foxf2 leads to sustained epidermal growth factor receptor (EGFR) activation. Immunoblotting analysis of the phosphorylation status of EGFR and total EGFR protein levels in shFoxf2- and shCtrl-expressing NMuMG cells treated with transforming growth factor (TGF)β for the times indicated. Actin was used as a loading control. b–d NMuMG cells expressing shRNA specific for Foxf2 or control shRNA were treated with TGFβ and AG1478 (EGFR inhibitor (EGFRi)) or control solvent (dimethyl sulfoxide (DMSO)) for the indicated times. b Cell numbers were determined using a Neubauer chamber. c EGFR inhibition significantly increases apoptosis in Foxf2 knockdown cells. Apoptosis was detected by Annexin-V staining and flow cytometry. d Treatment with AG1478 decreases EGFR activation in shFoxf2 cells to a similar extent as seen in TGFβ-treated NMuMG cells expressing control shRNA. Immunoblotting analysis for EGFR phosphorylation and total EGFR levels is shown. Tubulin was used as a loading control. e Foxf2 regulates the expression of EGFR ligands by direct transcriptional repression. Chromatin immunoprecipitation of Foxf2 was performed either on HA-Foxf2 expressing or control NMuMG cells treated for 2 days with TGFβ. Immunoprecipitated DNA fragments were quantified by quantitative PCR using primers covering base pairs −450 to −253 of the Btc promoter region, base pairs −851 to −654 of the Ereg promoter, and base pairs +1086 to 1210 of the Areg exon 2. Enrichment (IP/input) for specific primers was calculated relative to primers covering an intergenic region. f Individual depletion of Btc or combined depletion of betacellulin (Btc), epiregulin (Ereg), and amphiregulin (Areg) reduces cell numbers in shFoxf2- but not in shCtrl-expressing NMuMG cells in the presence of TGFβ for 4 days. Cell numbers were determined using a Neubauer chamber. Data are shown as mean ± SEM of at least three independent experiments. Statistical values were calculated using a paired/unpaired two-tailed t test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001