Vesicle trafficking in phosphoinositide phosphatase-knockdown cells

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A series of RAW264.7 macrophages was cloned that stably express the shRNA targeting the specific one of the phosphoinositide phosphatase isozymes. The targeted enzymes were eight 3'-phosphatases (PTEN, Synaptojanin2, Sac1, Sac3, MTM1, MTMR2, MTMR9, MTMR14), one 4'-phosphatase (Inpp4a), and six 5'-phosphatases (SHIP1, SHIP2, Inpp5e, SKIP, OCRL, Inpp5b). Two lines of cells that express shRNA with different targeting sequence were prepared for each isozyme. The knockdown cells were examined for their ability to engulf IgG-opsonized particles. The amount and localization of phosphoinositide derivatives, PtdIns(3,4,5)P3, PtdIns(3,4)P2, and PtdIns(3)P, during the phagosome formation and maturation were also monitored. The results suggested several specific functions of the phosphatase isozymes.