Fig. 2. Fluid-phase endocytosis is reduced in cells depleted of Dyn2 protein using siRNA treatment. (A-C') Fluorescence micrographs of Clone 9 cells incubated with fluorescently conjugated dextran (A',B',C') following either mock (A,A') or Dyn2 siRNA (B-C') treatment. Dyn2 protein levels were analyzed by staining for Dyn2 using immunofluorescence (A,B,C). (A,A') No effect on fluid-phase endocytosis was observed when cells treated with transfection reagent alone for 72 hours were allowed to internalize fluorescently conjugated dextran for 60 minutes in low serum medium (0.2% FBS). (B,B') Cells transfected with a solution containing a pooled mixture of four distinct siRNA duplexes targeting Dyn2 and assayed 72 hours later, as for cells in A,A', showed a substantial reduction in Dyn2 staining (B) along with a marked reduction in the amount of dextran-positive cytoplasmic vesicles (B'). (C,C') Treatment of cells as for B,B' but in the presence of high serum medium (10% FBS): a more modest reduction in dextran internalization was observed than in low serum conditions. (*) Indicates a cell with reduced Dyn2 levels. (D) Quantitation of the amount of fluorescently conjugated dextran internalized by Clone 9 cells that had either been mock-treated for 72 hours, treated with a pooled mixture of Dyn2 siRNA duplexes for 72 or 96 hours, or treated with Dyn2 siRNA duplex #2 for either 72 or 96 hours, in the presence of low serum (black bars) or high serum (white bars) medium. Fluid-phase endocytosis was reduced in cells treated with either the pooled mixture of siRNA duplexes (Pool) or siRNA duplex #2 (#2), as compared with mock-treated cells, under both low serum (0.2% FBS) and high serum (10% FBS) conditions; however, the effect was more dramatic under lower serum conditions. Values obtained from siRNA-treated cells were normalized to those of mock-treated cells under similar serum conditions. Results represent the average ± s.d. of 50 cells measured in each of three independent experiments. Bar, 10 µm.