Fig. 2.

Coactivation of gene expression by EGFR and RHA. (A) Costimulation of cyclin D1 (pCCD1-Luc, Left) and iNOS (piNOS-Luc, Right) promoter activity by EGFR and RHA. P values calculated from Student's t test are shown above paired bars. (B) Knockdown of RHA expression diminishes EGFR-induced promoter activity. HeLa cells with stable expression of indicated shRNAs were transfected with plasmids. Luciferase assay was performed after 5 h EGF stimulation. The expression levels of EGFR and RHA are shown in the lower panel. The density of the RHA band was quantified using ImageJ with the density of the basal level band (i.e., lane 1 without EGF) from control shRNA set as 100. The numbers under other bands are the relative band densities as compared with the density of lane 1. Ctrl, control; IB, immunoblotting. (C) ATRS-dependent activation of the cyclin D1 promoter by EGFR and RHA. Relative luciferase activities (i.e., percentage of wild-type ATRS promoter activity) are presented as the mean results ± SD (n = 3). (D) Association of EGFR/RHA with the cyclin D1 gene promoter. (Top) Binding of EGFR and RHA to the cyclin D1 gene promoter in A431 cells. The band's density was quantified using ImageJ with the band density in lane 3 set as 1. The numbers under other bands are the relative densities as compared with the density of the band in lane 3. (Middle) Sequential ChIP-PCR analysis of the association of EGFR and RHA with the cyclin D1 promoter. (Bottom) Reduced binding of EGFR to the cyclin D1 promoter after RHA knockdown. (E) Reduced association of EGFR with the iNOS promoter in A431 cells after RNA knockdown. (Upper) Normal ChIP-PCR. (Lower) Quantitative ChIP-PCR.