Effect of RNAi-mediated knock-down of class III PI3K expression on ROS responses to S aureus and E coli in GFP-PX-RAW cells. GFP-PX-RAW cells were infected with retroviral constructs directing expression of nonsilencing control shRNAi (CON), or 2 independent class III PI3K shRNAi constructs (Vps34.1, Vps34.2), as described in “RNAi knockdown of class III PI3K.” (A) shRNAi-treated GFP-PX-RAW cells were harvested, sonicated into SDS sample buffer, subjected to SDS-PAGE, and immunoblotted for class III PI3K and, as a loading control, actin. (B) Control (♦) and 2 Vps34 (□ and ▵) shRNAi GFP-PX-RAW cell populations were harvested, incubated with luminol, and ROS generation measured over time in response to serum-opsonized S aureus (i) or E coli (ii) as described in Figure 1. Data shown are mean plus or minus range for duplicate measurements in one representative experiment of 3. (iii) Total accumulated superoxide production for control and Vps34shRNAi GFP-PX-RAW cells in response to S aureus and E coli from 4 independently derived cell populations, was calculated over a 20-minute measurement period. Data are mean plus or minus SEM.