JCS010686 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Anti-Dyn2 antibodies inhibit fluid-phase endocytosis in multiple epithelial cell types. (A-F) Fluorescence micrographs of mouse hepatocytes (A,B), HeLa cells (C,D) and MDCK cells (E,F) microinjected with either anti-Dyn2 (A,C,E) or anti-pan-dynamin (MC65; B,D,F) antibodies as in Fig. 1 of the main text and then incubated with either fluorescently conjugated dextran (A-F) or transferrin (not shown) 4-6 hours following injection. Regardless of cell type, anti-dynamin antibody-injected cells (*) showed a reduction in dextran internalization as compared to the surrounding uninjected cells. Bar, 10 μm. (G) Quantitation of the inhibitory effects of antibody injection based on scoring of antibody-injected mouse hepatocytes and HeLa cells as either positive or negative for fluorescently conjugated transferrin (white bars) or dextran (black bars). Microinjection of control antibodies (anti-kinesin, heat-inactivated anti-dynamin, or anti-caveolin-1 antibodies) did not inhibit transferrin or dextran internalization in either cell type, whereas microinjection of anti-dynamin antibodies (MC65 and anti-Dyn2) did inhibit internalization of these ligands in both cell types.

• Supplemental Figure S2 -

  Fig. S2. Dyn2 siRNA treatment of epithelial cells reduces transferrin internalization. (A) Cartoon of the domain structure of the Dyn2 protein and the corresponding regions to which siRNA oligonucleotides were made. Western blot analysis of lysates from mock-treated Clone 9 cells, cells treated with a pooled mixture of four Dyn2 siRNA duplexes, or cells treated with each of the Dyn2 siRNA duplexes individually using anti-Dyn2 antibodies indicated a varying degree of reduction in Dyn2 protein levels depending on the siRNA duplex used, with siRNA duplex #2 being the most efficacious. (B-C′) Fluorescence micrographs of Clone 9 cells that were treated with transfection reagent alone (Mock; B,B′) or a pooled mixture of Dyn2 siRNA duplexes (C,C′) and then immunostained for Dyn2 (B,C) after incubation with fluorescently conjugated transferrin (B′,C′). Mock-treated cells exhibited a normal Dyn2 staining pattern (B) and transferrin uptake did not appear altered (B′). By contrast, clusters of cells showing reduced Dyn2 protein levels after treatment with the pooled mixture of Dyn2 siRNA duplexes (C) displayed a corresponding reduction in the internalization of transferrin (C′). (*) Indicates cell with reduced Dyn2 levels. Bar, 10 μm. (D) Quantitation of internalized transferrin in mock-treated cells, cells treated with a pooled mixture of Dyn2 siRNA duplexes for 72 hours or 96 hours, or cells treated with Dyn2 siRNA duplex #2 for 72 hours based on fluorescence intensity measurements. Mock treatment of cells did not result in a reduction in transferrin internalization; however, treatment of cells with either the pooled mixture of Dyn2 siRNA duplexes or Dyn2 siRNA duplex #2 reduced transferrin internalization by ∼70-80%. Results represent the average ± s.d. of &γτ;50 cells measured in each of three independent experiments.

• Supplemental Figure S3 -

  Fig. S3. Reduction of either caveolin-1 or clathrin protein levels does not inhibit dextran uptake. (A-C′,E-G′) Fluorescence micrographs of Clone 9 cells treated with siRNA for 72 hours to reduce either caveolin-1 (A-C′) or clathrin (E-G′) protein levels. Following treatment with siRNA for 72 hours, cells were challenged to internalize fluorescently conjugated cholera toxin B (A′), transferrin (E′) or dextran (b′,c′,f′,g′), fixed, and immunostained for either caveolin-1 (A-C) or clathrin (E-G). Caveolin-1 siRNA treatment reduced the levels of caveolin-1 present in some cells (A-C, asterisks), and this correlated with a reduced amount of internalization of cholera toxin B (A′). However, reduction in caveolin-1 protein had no inhibitory effects on the internalization of dextran (B′,C′). Similarly, clathrin siRNA treatment reduced the protein levels of clathrin in some cells (E-G, asterisks), and this correlated with a reduction in transferrin internalization (E′). Reduction in clathrin protein levels did not, however, affect dextran internalization (f′,g′). (d,d′,h,h′) Quantitation of siRNA-treated cells based on fluorescence intensity measurements indicated an ∼70% decrease in the internalization of cholera toxin B (in caveolin-1 siRNA-treated cells, D) or transferrin (in clathrin siRNA-treated cells, H), but no reduction in dextran internalization in either case (D′,H′). Mock treatment of cells was used as a control for all experiments. Results represent the average ± s.d. of &γτ;50 cells measured in each of two independent experiments.