Figure 3. Characterization of EndoIRKO-Min mice.

A–B. Cdh5-cre Apc^Min/+ mice were crossed with mT/mG mice, a double-fluorescent reporter of cre recombinase activity. Offspring have ubiquitous expression of a red fluorescent protein, i. e. membrane-targeted tandem dTomato (dTomato), except in cells with cre-mediated recombination of the mT/mG transgene where dTomato expression is replaced with expression of membrane-targeted enhanced green fluorescent protein (GFP). 80 μm cryosections of formalin-fixed tissue was stained with DAPI only and imaged with confocal microscopy. Scale bar, 50 μm. A. Normal intestine. B. Polyp. C–E. Tumors from 3 EndoIRKO-Min mice and 3 controls were enzymatically digested and cells sorted by FACS using antibodies against EpCAM or CD31; status as double-negative or single-positive for this antigens is indicated below graphs. mRNA expression was measured by real-time PCR and normalized to expression of Rplp0, a ribosomal RNA. C. Enrichment of epithelial cells in EpCAM+ CD31− fraction shown by expression of Epcam mRNA. D. Enrichment of endothelial cells EpCAM− CD31+ fraction shown by expression of Kdr mRNA. Please note log scale. E. Expression of Insr mRNA. F. Tumor cells from Cdh5-cre Insr^lox/lox Apc^Min/+ mice (EndoIRKO-Min, “E”) or their littermate Insr^lox/lox Apc^Min/+ controls (“c”) were grown in a mixed culture and endothelial cells isolated by magnetic selection using ICAM-2 conjugated Dynabeads at the first and second passage. ICAM-2-negative cells were collected at the first of these immuno-magnetic sortings while tumor endothelial cells were used after passage 3. Bone marrow, peripheral blood leukocytes and spleen were isolated and lysed. Representative Western blots of lysate is shown. G. Quantitation based on densitometry of Western blots using material from 3 pairs of animals. H. Isolated tumor endothelial cells were serum-starved overnight, then treated with 10 nM insulin for 5 minutes. Representative Western blots are shown. I–J. Quantitation based on lysate from 4 independent experiments. K. glucose tolerance test. Plasma glucose concentrations were measured after intraperitoneal injection of 2 mg/g glucose in 5 EndoIRKO-Min mice and 5 control animals. L. Insulin tolerance test. Plasma glucose concentrations after intraperitoneal injection of 0.75 mU/g Humulin R in 5 EndoIRKO-Min mice and 5 control animals. All panels: *, p<0.05.