Figure 2

ISRE GG->CC mutant impairs GFP expression in CEACAM1 promoter-driven constructs. (A) Schematic diagram of the -1,135-kb fragment, in relation to the ATG start codon, of the CEACAM1 promoter (upper black arrow) fused in translational reading frame to GFP (green box). Known transcription factor binding sites to IRF-1, SP1 and USF-1 are indicated. A mCherry reporter that was co-transfected and used as a quantitation control was fused to CEACAM1 WT ISRE (lower black arrow). (B) Nucleotide comparisons of the 21-bp ISRE with mutant alleles (A->T and GG->CC). (C) RT-PCR of GFP gene expression. Total RNAs were isolated, subjected to semi-quantitative PCR and separated by electrophoresis on a 1% agarose gel. GFP and GAPDH are indicated to the right and primer positions for ISRE promoter-driven GFP are indicated in the schematic. (D) Flow cytometric analyses of CEACAM1 ISRE WT and mutant promoter alleles GFP reporter constructs after Ad-null or Ad-IRF-1 treatment showing mCherry (Y-axis) and GFP (X-axis). Results are from one of three experiments with similar results. (E) Histogram of Quadrant 2 (Q2) in Figure 2D shows % cells expressing vector, ISRE WT, or mutant ISREs in GFP and ISRE WT mCherry reporter activity (% GFP⁺ mCherry⁺ cells). Results are from n = 3 independent experiments and **, p < 0.01 versus ISRE WT controls are indicated.