Figure 9.

Effects of knockdown of the individual PI 4-kinases on [³²P]phosphate labeling of phosphoinositides and their response to AngII stimulation in HEK-293-AT1 cells. HEK-293 cells stably transfected with the AT1a receptors were treated with siRNA for 3 d as described under Materials and Methods. The extent of knockdown was determined by Western blot analysis (A). On the fourth day, cells were labeled with [³²P]phosphate for 3 h in phosphate-free medium. Cells were then either treated with AngII (10⁻⁷ M) or saline and incubated for an additional 10 min. Reactions were terminated by PCA, and lipids were extracted from the cell pellets, separated by TLC, and analyzed by a PhosphorImager (B). Due to cell loss in the PI4K-depleted cells, the individual PtdIns4P and PtdIns(4,5)P₂ spots were normalized to the PtdA/PtdIns values measured in unstimulated samples of the respective groups. These ratios were then compared between the PI4K down-regulated or control siRNA-treated cells in each of the three experiments performed in duplicates (C). In each experiments, the effect of 250 nM PIK93 was also determined in the AngII group. Because PIK93-treated cells showed no difference compared with those treated with AngII only, these values were pooled in the statistical analysis. Means ± SEM are shown, and the p values obtained in one-sample t tests compared with control siRNA-treated cells are shown above the bars.