Fig. 1. Wortmannin enlarges EGFR-containing endosomes by a PI3K-independent pathway. (A) Effects of the addition of PI3K reaction products on wortmannin-induced enlargement of EGFR-containing endosomes. BT20 cells were treated with wortmannin and a mixture of three PI3K reaction products including PtdIns-3-P, PtdIns-3,4-P₂ and PtdIns-3,4,5-P₃, and stimulated with EGF for 30 min or not stimulated. EGFR (left panel and arrows) and phospho-Akt (right panel and arrowheads) localization were determined by indirect immunofluorescence. (B) Effects of addition of wortmannin and LY294002 on the size of EGFR-containing endosomes in MDCK cells. Left panel, cells treated with wortmannin. Right panel, cells treated with LY294002. Cells were treated with wortmannin or LY294002 at the indicated concentrations for 15 min and then stimulated with EGF (100 ng/ml) for 30 min. EGFR (arrows) was detected by indirect immunofluorescence. Size bar = 20 µm. (C) Quantification of the results from (B).