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( a– d) Streptozotocin (STZ)-treated mice were compared with their vehicle-treated controls (Veh) and ( e– h) ob/ob mice were compared with their wild-type controls (Con) all mice were killed in the non-fasted state. ( a– h) Eight- to ten–week-old male mice were killed in the non-fasted state. Hepatic FoxO1 protein ( s) was measured by western blotting whole-cell lysates. Hepatic gene expression ( q, r, t) was measured by real-time PCR. ( q– t) Eight- to ten-week-old male LIRKO mice were fed a chow diet with or without supplementation of lovastatin and ezetimibe (L+E) for 1 week and euthanized in the non-fasted state. Representative images are shown in l and the left panels of o, p. of three independent experiments * P<0.05 (Student's t-test). ( o, p) Data represent the mean and s.e.m. ( l, o, p) Whole-cell lysates were subjected to western blotting ( l, o, p) and quantification ( o, p right panels).
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* P<0.05 (Student's t-test) n=3 wells per condition results are representative of three independent experiments. Gene expression was measured by real-time PCR ( m, n). Alternatively, shRNA expression plasmids were transfected into H2.35 hepatoma cells ( p). ( l– o) Primary mouse hepatocytes were infected with control adenovirus or adenovirus expressing SREBP-2 ( l– n) or miR-182 ( o). n=5–7 * P<0.05 (Student's t-test) LIRKO versus Flox mice treated with the control ASO # P<0.05 (Student's t-test) control versus FMO3 ASO treatment of LIRKO mice. Glucose ( d) and insulin ( e) tolerance testing were performed after 5 weeks of ASO treatment. ( c) TMAO was measured in plasma collected at the time of sacrifice using LC/MS. Protein levels were measured by western blotting whole-cell lysates ( b, g) or nuclear fractions ( i). The data are row-normalized with red and blue representing high and low expression, respectively. Hepatic gene expression ( a, f, h, i, j, k) was measured by real-time PCR, and in ( h) expressed as a heat map, with each column representing data from a single mouse. ( a– k) Four- to six-week-old male Flox and LIRKO mice were treated with control (Con) or FMO3 ASO for 7 weeks and killed in the non-fasted state.