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. 2015 Dec 21;6:8995. doi: 10.1038/ncomms9995

Figure 2. Reduced atherosclerosis in M4K4 ECKO mice.

Figure 2

Map4k4 flox/flox animals were crossed with Cdh5(PAC)-ERT2-Cre animals and injected with tamoxifen for 5 consecutive days at 6–8 weeks of age. (a) Cre-mediated Map4k4 exon-7 deletion. (b) Schematic of injection and feeding scheme. (c) Messenger RNA was extracted and qRT–PCR was performed for Map4k4 in primary MLECs, the unselected, non-EC fraction of mouse lung cells and peripheral blood leukocytes. The data represent the mean±s.e.m. as normalized to 36b4 expression (*P<0.05, N=6–8). (d) Immunoblots were performed for Map4k4 or tubulin in immune-selected primary MLECs and the unselected, non-EC fraction of mouse lung cells. Western blots are representative of 6–8 animals per group. (e–j) Flox/flox and MAP4K4 ECKO mice were crossed with Apoe−/− mice and fed a WD for 16 weeks as in b. (e) Left, Oil Red-O-stained en face aortic preparations from flox/flox and MAP4K4 ECKO animals. Right, quantification of Oil Red-O-stained area. Data represent the mean±s.e.m. (***P<0.0005, N=8–10). (f–j) Aortic root sections of flox/flox and MAP4K4 ECKO Apoe−/− animals stained with (f) haematoxylin and eosin (H&E) (scale bar, 250 μm), (g) Oil Red-O (scale bar, 250 μm), (h) trichrome (scale bar, 250 μm), (i) smooth muscle actin (scale bar, 250 μm (top image); scale bar, 100 μm (bottom image)) and (j) Cd68 (scale bar, 250 μm (top image); scale bar, 100 μm (bottom image)). Left panel, representative images. Right panels, quantification of stained area or as a percentage of lesion area. Data represent the mean±s.e.m. (*P<0.05, **P<0.005, ***P<0.0005, N=8–11). DAPI, 4,6-diamidino-2-phenylindole.