Figure 3

Generation of metal and IFN-responsive reporter PCR products and their performance. (A) HEK293 cells in 96-well plates were transfected with 75 ng of purified PCR products generated from reporter vector using the Forward primer that contains MRE sites (Table 1, SEQ. 4 and 5). After 20 hr, the cells were treated with 10 μM cadmium for 16 hrs. Data is Mean ± SEM (quadruplicate) – a representative experiment of two- of fold increase due to cadmium in GFP fluorescence levels that were normalized to background fluorescence from non-MRE PCR product. (B) Huh7 cells in 96-well plates were transfected with 75 ng of purified PCR products in which the Forward primer includes two putative ISRE sites (Table 1 SEQ 6). After approximately 20 hr, the cells were treated with IFN-α (log_0.5) for an additional 16 hrs. Right panel: An IFN-resistant HEK293 cells were transfected with the ISRE-containing EGFP PCR product (20 hr) and then treated with IFN-α (33 IU/ml) for 16 hr. Data are from one experiment (Mean ± SEM (n = 4). (C) Dose-response curve for IFN action on GFP reporter activity from the ISRE-containing PCR product (normalized to fluorescence levels from non-responsive reporter). *, p < 0.01, **p < 0.005 and ***p < 0.0001. (D) Total RNA was extracted from Huh7 cells that were transfected with IFN-responsive reporter PCR products in the presence or absence of IFN-α (100 U/ml) for 4 or 6 hrs. RT-PCR was performed using primers specific to EGFP as described in Methods. PCR products were run on gel, and β-actin normalized signal intensities of ethidium bromide-stained products (Mean ± SEM (n = 3) were quantitated using AlphaEase.