Fig. 1. siRNA-mediated suppression of hVps34 expression in U-251 glioma cells induces accumulation of LAMP1-positive cytoplasmic vacuoles. (A) U-251 cells infected with retrovirus carrying the control or hVps34 KD siRNA sequences surviving after 5 days of puromycin selection were subjected to immunoblot analysis with a polyclonal anti-hVps34 IgG as described in the Materials and Methods. Nuclear lamin B₂ served as one of several controls for nonspecific effects of the siRNA. (B) Phase contrast images of the live control and hVps34 KD cells. Bar, 10 µm. Based on counting 100 cells in multiple phase micrographs, 73% of the hVps34 KD cells exhibited the vacuolar phenotype, defined as multiple small-sized to intermediate-sized vacuoles (0.5-1.0 µ), with at least two vacuoles per cell exceeding a diameter of 2 µ. (C) Control and hVps34 KD cells were seeded at 100,000 cells/dish in 35 mm dishes. 24 hours later, cells were examined by immunofluorescence microscopy using a primary antibody against the lysosomal and late endosomal membrane protein LAMP1. Bar, 10 µm.