Fig. 9. Bead uptake and d/A surface redistribution in DCs exposed to PIP3. Panels A-D are merged images of DCs exposed to phosphoinositides for 20 minutes, loaded or not with the Bds for 10 minutes, fixed and surface immunolabelled for d/A. With PIP3, Bd uptake and d/A surface appearance took place even after pretreatment with Wort (A) or preloading with BAPTA (B). Notice the large size of the PIP3-induced Bd-rich puncta mostly located near the plasma membrane (tridimensional reconstruction in supplementary material Fig. S5); (compare with the cells exposed to the beads only: Fig. 1D,E; Fig. 5A, Fig. 8D,F,H). In PIP3-exposed DCs pretreated with Wort (not shown) or preloaded with BAPTA (C) the d/A surface redistribution occurred even without Bd loading. By contrast, the PIP3 precursor, PIP2, had no effect in DCs, not-pretreated and pretreated with Wort (not shown) or with BAPTA (D). Panels E-G show merged images of DCs pretreated with PIP3 as in A-C and then loaded for 10 minutes with Bds (green) together with FM4-64 (FM, red). The large Bd-rich puncta of E were sealed, discrete vacuoles because after washing they retained their FM staining. Some loss of the FM signal was visible in contrast to DCs pretreated with Wort (F) and, even more, in those preloaded with BAPTA (G). The Bd-rich puncta induced by PIP3 were negative for EEA1 (red in H). Bar, 10 µm (C), also valid for A,B,D,H. Bar, 10 µm (E), also valid for F and G.