Fig. 4. siRNA-mediated suppression of endogenous PIKfyve leads to formation of enlarged, swollen cytoplasmic vacuoles. Five siRNA duplexes (I-V) were designed, each targeting a distinct region of human PIKfyve (see supplementary material Table 1). (A) HeLa cells were transiently transfected with each individual siRNA or a combination of siRNA duplexes II and V, for 72 hours prior to determining the level of PIKfyve using an antibody against human PIKfyve. Tubulin was used as a loading control. Depending on the siRNA used, varying levels of suppression were achieved, with the highest suppression routinely obtained when using a combination of siRNAs II and V (ratio 1:1). Data are representative of one series of experiments typical of at least three other independent experiments. (B) HeLa cells were transiently transfected with either control siRNA, PIKfyve-specific siRNA duplex II or a combination of PIKfyve-specific siRNA duplexes II and V. After 72 hours, cells were fixed and analysed as phase-contrast images. Each image is of a random field of view. Arrowheads and arrows indicate small and large vacuoles, respectively. Bars, 45 µm. (C) Quantification of the percentage of cells displaying a vacuolar phenotype was determined by visual inspection. A cell was scored as having such a phenotype if two or more swollen vacuoles were visible. Data are from 100 cells imaged for each condition.