Fig. 3. Disruption of Dyn2 function in epithelial cells stimulated with EGF does not prevent macropinocytic internalization of fluid. (A-B') Fluorescence micrographs of HeLa (A,A') or Clone 9 (B,B') cells treated with Dyn2 siRNA for 72-96 hours prior to incubation with fluorescently conjugated dextran for 30 minutes in low serum medium (A,B) or low serum medium plus 30 ng/ml EGF (A',B'). Cells with reduced Dyn2 levels (*) showed a marked reduction in fluid-phase endocytosis under unstimulated conditions, as compared with surrounding untransfected cells (A,B). Treatment of cells with EGF, however, induced a significant internalization of fluorescently conjugated dextran via the macropinocytic pathway, regardless of Dyn2 expression levels (A',B'). (C-D') Fluorescence micrographs of HeLa (C,C') or Clone 9 (D,D') cells microinjected with anti-Dyn2 antibodies as described in Fig. 1 and allowed to internalize fluorescently conjugated dextran under low serum conditions for 60 minutes in the absence (C,D) or presence (C',D') of EGF. As seen for Dyn2-siRNA-treated cells, dextran uptake was noticeably reduced in antibody-injected cells under low serum conditions (C,D), as compared with controls, whereas this effect was more modest in cells treated with EGF (C',D'). (E,F) Quantitation of internalized fluorescently conjugated dextran in Clone 9 (black bars) and HeLa (white bars) cells treated with Dyn2 siRNA (E) or microinjected with anti-Dyn2 antibodies (F) based on fluorescence intensity measurements. Values obtained from siRNA-treated cells were normalized to those of mock-treated cells under similar conditions, and values obtained from microinjected cells were normalized to those of surrounding uninjected cells. Results represent the average ± s.d. of 50 cells measured in each of three independent experiments. Bar, 10 µm.