Reporter gene assay for transcription factors influenced by EGFR-TKIs.

<p>(<b>a</b>) Luciferase reporter assay. The pGL4 vectors, which encode response elements for transcription factors (indicated) or a minimal promoter (minP; a negative control) linked with the <i>Photinus luciferase</i> gene, were transfected together with phRL-TK carrying the <i>Renilla luciferase</i> gene (as a reference) into PC-9 cells. After 24h-incubation, the cells were exposed to 10μM gefitinib or DMSO (a vehicle) for 6h and subjected to a dual-luciferase assay. The activity of the <i>Photinus</i> luciferase (<i>P</i> luc.) was normalized to that of the <i>Renilla</i> luciferase (<i>R</i> luc.). Data are shown as mean ± standard deviation (SD) (<i>n</i> = 4). Significant difference between gefitinib and DMSO was examined by means of two-tailed Student’s t-test (*<0.05, **p<0.01, ***p<0.001). (<b>b</b>, <b>c</b>) Effects of gefitinib or erlotinib on transcription factors. The GL4-plasmid carrying the NF-κB response element (<b>b</b>) or the serum response element (SRE) (<b>c</b>) was co-transfected with phRL-TK into PC-9 cells, and after 24h-incubation, the cells were treated with 0, 0.1, 1.0 and 10μM of gefitinib or erlotinib for 6h. Dual-luciferase assay followed by normalization were carried out as in <b>a</b>. Normalized data were further normalized to the data obtained from cells treated with no EGFR-TKIs (0 μM) as 1. Data are shown as mean ± standard deviation (SD) (<i>n</i> = 3).