Effect of RNAi-mediated knock-down of class III PI3K expression on endosomal and phagosomal PtdIns(3)P accumulation. GFP-PX-RAW cells expressing nonsilencing control (CON) or independent class III PI3K (Vps34.1, Vps34.2) shRNAi were adhered to glass coverslips and incubated in the absence (A) or presence (B) of RITC-labeled, serum-opsonized S aureus as described in “RNAi knockdown of class III PI3K.” Samples were fixed, mounted, and GFP-positive endosomes (A) and phagosomes (B) visualized on a Zeiss LSM 510 META point-scanning microscope. Shown are images from a single 1 μm confocal plane. (C) GFP-endosomal and cytosolic accumulation (i) and GFP-positive phagosomes (ii) from cells described in panels A and B, respectively, were quantified from 6 × 1 μm z-section confocal images using LSM 510 software. Data are mean plus or minus SEM for at least 20 cells, or 100 phagocyte events in control (■) and Vps34.1 (), Vps34.2 () shRNAi-treated cells, and are expressed as endosomal GFP accumulation as a ratio of cytosolic GFP accumulation in subpanel i or GFP-positive phagosomes expressed as a percentage of total phagosomes in subpanel ii.