Egr2 contains an IRES element. (A) Scheme of bicistronic constructs used for reporter assays. (B) Bicistronic reporter plasmids were cotransfected with SV40-β-Gal plasmid into MCF7 cells. Twenty-four hours after transfection, renilla and firefly activities were measured and normalized to β-galactosidase activity. Data are presented as means ± SEM (n > 3). (**) P < 0.01 relative to phpRF. (C) In vitro transcribed mRNAs of the indicated reporter plasmids were transfected into MCF7 cells. Twenty-four hours after transfection, renilla and firefly activities were measured. Data are presented as means ± SEM (n > 3). (**) P < 0.01 relative to hpRF. (D) MCF7 cells were cotransfected with the indicated reporter constructs and SV40-renilla plasmid. Forty-eight hours after transfection, firefly activity was measured and normalized to renilla activity. Data are presented as means ± SEM (n > 3). (E) RNA isolated from cells transfected with phpRF or phpR-egr2-F was DNAse-treated. cDNA was synthesized, and PCR was performed with specific primers to amplify full-length RL or R-egr2-L mRNAs. PCR products were visualized via agarose gel electrophoresis. Data are representative for at least three independent experiments. (F) RT-qPCR analysis of the amount of firefly mRNA normalized to renilla mRNA. Data are presented as means ± SEM (n = 3). (G) MCF7 cells were transfected with phpR-egr2-F and treated with CM or Ctr for 24 h. IRES activity was calculated as the ratio of firefly to renilla activities and is given relative to Ctr. Data are presented as means ± SEM (n > 3). (**) P < 0.01.
