Angptl4 gene transcription is mediated by the ERK/c-Myc pathway in LN229-vIII cells. (A) Inhibition of Angptl4 mRNA expression by treatment with U0126. LN229-vIII cells were treated with U0126 or gefitinib for 24 h. Total RNA was extracted and reverse- transcribed, followed by real-time PCR. (B) EMSA assay for Myc/MAX using a nuclear extract of LN229 cells transfected with mock, wtEGFR and EGFRvIII. (C) LN229-vIII cells were treated with U0126 (0.1, 1, 10 μM) for 24 h; thereafter, the nuclear proteins were extracted and used in the EMSA assay. Complex, the specific DNA-protein (probe/c-Myc) complex. (D) Knockdown of c-Myc in the LN229-vIII cells was accomplished using siRNA. Angptl4 (black bar) and c-Myc (white bar) mRNA expressions were determined by real-time PCR and normalized to the expression level of β-actin. Bars show the mean ± SEM (n=4). (E) Formalin-fixed chromatin-protein complexes were sonicated, immunoprecipitated for c-Myc, eluted, and quantitative PCR for the promoter of Angptl4 was performed. The enrichment was calculated in fold-values by normalization to the negative control IgG and input control. Bars show the means ± SEM (n=3). Significant differences are shown: ** p<0.01, *** p<0.001.