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Fig. 3. The insulin-induced increase in the formation of lipid droplets is dependent on PLD1 and ERK2. (A) NIH 3T3 cells were transfected with PLD1 siRNA or control siRNA (as indicated). After 2 days the cells were incubated with 3 nM insulin for 2 hours and the total area of Oil Red O-stained lipid droplets/cell was determined (from all cells in 50 randomly selected micrographs). The total area of Oil Red O-stained lipid droplets/cell in the cells treated with insulin and control siRNA was set to 100% and the recovery of lipid droplets in the other two groups were related to this group (mean ± s.d.; n=3. 
P=0.002 vs Insulin+PLD1 siRNA and P=0.004 vs Buffer+Control siRNA; 
NS vs Buffer+Control siRNA; one-way ANOVA). (B) NIH 3T3 were incubated with 25 µM Ste-Mek113 for 1 hours and then with 3 nM insulin together with 25 µM Ste-Mek113 for 2 hours. The total area of Oil Red O-stained lipid droplets/cell were analyzed as in A and the insulin-treated cells were set at 100% (mean ± s.d.; n=5. P=0.036 vs Insulin+Ste-Mek113 and P=0.002 vs control; NS vs control; one-way ANOVA).
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