Fig. 1. PLD1, but not PLD2, promotes the formation of lipid droplets in NIH 3T3 cells. (A,B) Effect of transient transfection of NIH 3T3 cells with wild-type PLD1 (PLD1wt), K898R PLD1 (a catalytically inactive mutant) or an empty vector on the area of Oil Red O-stained lipid droplets per cell. (A) One day after the transfection with either PLD1 (left), the catalytically inactive mutant (right) or an empty vector (not shown), the cells were stained with Oil Red O to stain the lipid droplets. Bar, 10 µm. (B) BioPix software was used to calculate the total area of Oil Red O-stained droplets per cell. Values are mean ± s.d. of all cells in 20 randomly selected micrographs from each group (^*P<0.001 PLD1wt vs K898R PLD1 and empty vector; Not significant (NS) K898R PLD1 vs empty vector, one-way ANOVA). (C) Accumulation of radioactive triglycerides after incubation with [³H]palmitic acid for the indicated times in NIH 3T3 cells transiently transfected with wild-type PLD1 (triangles) or K898R PLD1 (squares). Values are mean ± s.d. from cells from three different culture dishes. (D) Influence of the transfection with PLD1 on the levels of ADRP in the cell. Cells were transfected as in A and the cell lysate was blotted against antibodies to ADRP. (E) Effect of transient transfection of NIH 3T3 cells with wild-type PLD2 (PLD2 wt), K758R PLD2 (a catalytically inactive mutant) or an empty vector on the area of Oil Red O-stained lipid droplets per cell (values are mean ± s.d. of all cells in 20 randomly selected micrographs from each group; NS). (F) The amount of ADRP as estimated by immunoblotting.