Fig. 2

The CDP-eth pathway is down regulated in PCYT2 knockdown cells. (A) PCYT2 mRNA (a) and protein (b, c) were diminished in PCYT2 knockdown (KD) cells. (B) Pulse experiments with [14C]Etn demonstrating incorporation into intermediates of the CDP-Etn pathway in control (WT) and KD cells. The cells were labeled with 1µCi [14C]Etn per dish for 1 and 2 h. The intracellular [14C]Etn and the synthesis of[14C]PEtn, [14C]CDP-Etn, and [14C]PE are shown. All measurements were performed in triplicate and expressed as means ± SEM from two separate experiments. Significant differences between WT and KD cells at different time points were determined with one-way ANOVA as indicated by * (P < 0.05) and ** (P < 0.005). C. Pulse-chase experiments using [14 C]Etn to demonstrate the degradation of metabolites of the CDP-Etn pathway. Cells were pulsed with 1 µCi [14 C]Etn for 2 h, washed, and then chased with cold ethanolamine for 0.5, 1, 2, and 3 h, as indicated. The turnover trends for [14 C]Etn, [14 C]PEtn, [14 C]CDP-Etn, and [14 C]PE are compared between WT and KD cells. The measurements were performed three times in duplicate experiments and expressed as means ± SEM. Significant differences between WT and KD cells at different time points were determined with one-way ANOVA as indicated by * (P < 0.05) and ** (P < 0.005).