Figure 4.

Dlx2 promotes resistance against TGFβ-mediated growth inhibition via activation of EGFR signalling. (A) Dlx2-expressing and control NMuMG cells were treated with TGFβ for 4 days in combination with the PI3K inhibitor ZSTK474 (0.239 μM) or DMSO (solvent control) and counted using a Neubauer chamber. (B) Dlx2-expressing and control NMuMG cells were treated with TGFβ for 4 days in combination with the MEK1/2 inhibitor PD98059 (9.35 μM) or DMSO (solvent control), and cell numbers were determined using a Neubauer cell counting chamber. (C) Dlx2 expression increases phosphorylation of the MAPK Erk1/2 as well as c-Myc total protein levels. Immunoblotting analysis of cell lysates from Dlx2-expressing and control NMuMG cells treated with or without TGFβ for 4 days. Immunoblotting against total Erk1/2 and GAPDH was used as loading control. (D) Dlx2 expression has no effect on the phosphorylation of PKB at Ser473, as determined by immunoblotting with an antibody specific for PKB phosphorylated at serine 473. Immunoblotting against total PKB and total-Erk1/2 was used as loading control. (E) Dlx2-expressing and control NMuMG cells were treated with TGFβ for 4 days in combination with the EGFR inhibitor AG1478 (3 μM) or DMSO (solvent control) and cell numbers were determined using a Neubauer chamber. (F) Dlx2 expression leads to increased phosphorylation of the EGFR at its activating tyrosine 1173. Immunoblotting analysis of cell lysates from Dlx2-expressing and control NMuMG cells with antibodies against pY1173-EGFR and total EGFR. Immunoblotting against vinculin was used as a loading control. Data are shown as mean±s.d. and are representative of at least three independent experiments. Statistical values are calculated by using an unpaired, two-tailed t-test ^**P⩽0.01; ^***P⩽0.001.