Vacuoles induced by MIPP are derived from macropinosomes that undergo progressive fusion events and accumulate at a pre-lysosomal stage. A) Time-lapse phase-contrast microscopy of U251 cells treated with MIPP. Images were captured at 30 sec intervals during the period between 13-80 min after the addition of 10 μM MIPP, and the images were assembled into a movie, which is available as Movie 1 (Additional file 1). The panels show sequential snapshots from the movie, with the elapsed time after addition of MIPP (min) indicated. Newly formed macropinosomes can be seen fusing with each other to form larger vacuoles. The small two-headed arrows point to vesicles that have fused in the subsequent frame. B) U251 cells were treated with 10 μM MIPP for 4 h, then incubated with the indicated fluorescent tracer or organelle marker, as described in Materials and Methods. The same field of cells is depicted in the matching phase-contrast and fluorescent images. In the top panel, the arrows indicate some of the specific vacuoles that have incorporated the Lucifer yellow. C) Cells were pretreated for 30 min with 12 μg/ml filipin or an equivalent volume of vehicle (DMSO), then 10 μM MIPP was added to the cultures. Phase-contrast images were acquired 100 min after the addition of the MIPP. D) Bafilomycin A1 (Baf-A) blocks the induction of vacuoles by MIPP. Cells were pretreated for 1 h in the presence (+) or absence (-) of 100 nM Baf A prior to addition of MIPP (+MIPP) or DMSO (-MIPP). Phase-contrast images were taken 1 h after addition of MIPP. The scale bars in all of the images represent 10 microns.