Figure 1

Forced expression by Ad-IRF-1 generates CEACAM1-L isoform in MDA-MB-468 breast carcinoma cells. (A) Model of cis and trans-acting elements involved in CEACAM1 AS. A general splicing mechanism involves physical interaction of hnRNP L and hnRNP A1 (enclosed in circles) with exon 7 (indicated by white box) at ESSs (indicated by – sign) to generate CEACAM1-S through repression of 3’ and 5’ ss (inhibitory arrows). hnRNP M promotes formation of CEACAM1-L through exon definition (arrows) as part of the regulatory splicing complex that participates with the Exon Splicing Enhancer (ESE; + sign). (B) Total cellular protein lysates from viral vector treated (Ad-null) or Ad-IRF-1 treated cells were subjected to Western blot and probed with antibodies to IRF-1, IRF-2, and p21. GAPDH was used as a loading control. (C) Induction of isoform CEACAM1-3L and CEACAM1-4L mRNA by Ad-IRF-1 after 24 h. (Upper) Total RNAs were isolated, subjected to RT-PCR and separated by electrophoresis on a 2.25% agarose gel. The position of CEACAM1-3 and CEACAM1-4 splice variants are indicated on right. M refers to a 100-bp DNA ladder (New England Biolabs). (Lower) Histogram quantification of data is n =3 independent experiments. The mean ± S.E. shown for percent exon 7 inclusion was calculated as the % [CEACAM1-L/(CEACAM1-L + CEACAM1-S)] mRNA. **, p < 0.01; ***, p < 0.001 versus Ad-null control. (D) Western blotting of cell lysates after induction of Ad-IRF-1 after 24 h with antibody T84.1 (red channel) which recognizes both isoforms of CEACAM1 (-S and –L) and antibody 229 (green channel) which recognizes CEACAM1-L isoform exclusively (each individually found in Additional file 3: Figure S3A-B). Shown is a composite of superimposed antibodies to highlight production of CEACAM1-L (yellow combined channels) after Ad-IRF-1 treatment.