EGR-1–induced expression in epithelial cell lines after contact with EGF transfectants. (A) After a 16-hour in vitro contact with stable transfectants (3 × 106 cells/well), 293 cells were harvested, total RNA was extracted and subjected to RT-PCR to quantify EGR-1 expression. 293 cells stimulated with EGF (50 ng/mL) for 1 hour were used as a positive control. (B) Quantitative analysis of the induction of EGR-1 expression was performed by imaging densitometry. The results are presented normalized relative to GAPDH expression. Results are representative of 3 independent experiments with several clones expressing EGF and controls. (C) pGL3-EGR-1 promoter construct (0.1 μg) was transiently cotransfected with pRL-TK Renilla luciferase (0.5 μg) into 293 cells as described in “Transient transfections and luciferase assays.” After 24 hours, the 293 cells were preincubated for 1 hour with blocking monoclonal antibodies specific for EGFR or control mouse IgG. Thereafter, the stable transfectants (3 × 106 cells/well) were added to adhrent cells for 16 hours. Cells were collected, and luciferase activity was measured. Controls included 293 cells without tumor cells. The firefly luciferase was normalized to Renilla luciferase activity. Results are representative of 3 independent experiments performed in triplicata. Bars represent SD; *P ≤ .05.
