Expression pattern of cytoplasmic isoforms of CEACAM1 in vivo . (A) Mean MDA-MB-468 tumor volumes (±SD) orthotopically implanted into mammary fat pads of NOD/SCID mice (n = 6). (B) Total RNAs were isolated from 6 tumors and two parental cell populations. RNAs were subjected to quantitative PCR using exon-junction specific primers. The % exon 7 inclusion was calculated by taking the CEACAM1-L/S ratio in tumor cells and comparing this fold increase to the CEACAM1-L/S ratio in parental cells. (C) Immunofluorescence staining of human CEACAM1 expressing cells from MDA-MB-468 tumor tissue. (Upper): Expression of CEACAM1-L but not CEACAM1-S is more intense at the invasive front of the breast carcinoma tissue. Immunofluorescence staining of human CEACAM1 expressing cells from MDA-MB-468 tumor tissue using antibodies directed to the ectodomain shown in red (5F4 mouse antibody 1:200; shown with white arrowheads) or the -L cytoplasmic tail of CEACAM1 shown in green (229 rabbit antibody 1:200; shown with white arrow) followed by secondary antibodies (goat anti-rabbit, Alexa Fluor 488 labeled and goat anti-mouse, Alexa Fluor 555 labeled; 1:200). For nuclei staining, Hoechst 33342 (blue) was used at final concentration of 1 μg/ml. Scale bar, 100 μm. (Lower): Secondary antibodies only were used as negative controls to rule out interference from auto-fluorescence.