FIPI (5-Fluoro-2-indolyl deschlorohalopemide) is a derivative of halopemide which potently inhibits both PLD1 (IC50 = 25 nM) and PLD2 (IC50 = 20 nM); prevents PLD regulation of F-actin cytoskeleton reorganization, cell spreading, and chemotaxis.

PLD2:20 nM, PLD1:25 nM

FIPI是一种强效的、浓度依赖性的PLD2抑制剂。研究表明，在标准体外实验条件下，FIPI对PLD1的抑制效果同样显著。将FIPI加入细胞培养基中，于进行体内PLD实验前1小时处理，发现FIPI是一种强效的PLD2抑制剂，IC50为10 nM。FIPI处理不影响PLD1向围绕核膜囊泡的典型定位和PLD2向质膜的定位，并且在过表达PLD1和PLD2的细胞中，使用增强型GFP融合的PIP2传感器评估后发现，FIPI不减少质膜上的PIP2可用性。FIPI未显著抑制用脂多糖刺激的骨髓衍生的巨噬细胞中的p38或ERK磷酸化。然而，FIPI抑制了PLD减少了通过fMLP引导的趋化性（p < 0.01），验证了PLD在调节趋化性中的作用，暗示PLD通过与影响MAKP信号传导不同的机制调控趋化性。

Phospholipase D activity is quantified using our established method of measuring the formation of [32P]-radiolabeled PBt. Cellular lipids are extracted and PBt is isolated using our published methods of lipid extraction and thin layer chromatographic separation, respectively. Radioactivity is measured using liquid scintillation counting and quantified as DPM normalized to 106 counts in the total cellular lipid extract or as percentage of control (vehicle-treated cells).

Cytotoxicity in MAECs is determined by assaying the extent of reduction in MTT in intact cells using the commercial MTT reduction assay kit. At the end of the experimental treatments, MTT solution (10% vol/vol in MEM) is added and the cells are incubated for 3 hours, following which MTT solvent is added in an amount equal to the original culture volume. Absorbance of the reduced MTT is determined spectrophotometrically, according to the manufacturer's recommendations.