tra12119-sup-0001-TableS1.docxWord 2007 document15KTable S1. MTT analysis of different human cancer cell lines after 72 h of incubation with Dyngo analogs. GI50 (μM) is the concentration that inhibits cell growth by 50% (the lower the value the greater the growth inhibition). Errors represent SEM (n = 3 independent experiments).
tra12119-sup-0002-AppendixS1.docxWord 2007 document213KAppendix S1. Dyngo library 2.
tra12119-sup-0003-SchemeS1.docxWord 2007 document673KScheme S1. Scheme for the synthesis of Dyngo analogs.
tra12119-sup-0004-FigureS1.docxWord 2007 document584KFigure S1. Dynasore is a poor dynamin I inhibitor when assayed in the presence of Tween-80. A) Structure of dynasore. B) Dose-dependent inhibition by dynasore of dynamin I GTPase activity stimulated by PS liposomes in the presence of Tween-80. C) IC50 values of dynamin I after activation by four mechanisms in the presence of Tween-80. Dynasore was either produced in house (synthesized), purchased from Sigma or obtained from the laboratory of Tom Kirchhausen (TK). Dynasore was tested at a range of concentrations up to a maximum of 1 mM, with the exception of data marked with *, which were tested up to 1.5 mM. D) Effect of dynasore on endocytosis of Tfn-A594 in U2OS cells. All data are means ± SEM of three independent experiments.
tra12119-sup-0005-FigureS2.docxWord 2007 document729KFigure S2. Dyngo compound 4a has no effect on dynamin binding to SH3 domains. Pull down of dynamin I in the absence or presence of the indicated 4a concentrations was performed using the SH3 domains of Grb2, endophilin I or amphiphysin I attached to GSH beads. The proteins were resolved on 12% SDS-PAGE gels and visualized using Coomasie Blue. The results are shown for one experiment performed in triplicate and the same results were obtained in two further independent experiments (in duplicate).
tra12119-sup-0006-FigureS3.docxWord 2007 document288KFigure S3. Dyngo compounds do not affect amphiphysin protein–protein interactions. The effect of dynasore and Dyngo compounds on binding of clathrin heavy-chain C-terminal domain or AP-2 alpha ear domain to amphiphysin 1 PRD + CLAP domains determined by ELISA assays. Data are mean and error bars represent SEM for triplicate measurements for n = 1.
tra12119-sup-0007-FigureS4.docxWord 2007 document509KFigure S4. Dyngo series 4a, 6a and dynasore are non-toxic and do not affect cell viability in HeLa cells. A and B) HeLa cells were exposed to MiTMAB or the indicated Dyngo compound for 8 h in the presence (A) and absence of serum (B) and then analyzed using an LDH assay. Data represent SEM (n = 2 independent experiments). C–F) Cell membrane integrity as an indicator of viability (C and E) and cell proliferation (D and F) in HeLa cells were analyzed after prolonged exposure (20 h) to 4a, 6a and dynasore in the presence (C and D) and absence of serum (E and F) using a trypan blue exclusion assay. Data represent SEM (n = 2 independent experiments).
tra12119-sup-0008-FigureS5.docxWord 2007 document387KFigure S5. Effect of dynasore analogs on mitochondria in HeLa cells. A) HeLa cells stably expressing H2B-mCherry (red) were serum-starved, incubated with Mitotracker Green FM (green) and imaged by confocal microscopy. The left panel shows cells at 40× magnification, while the right panel shows greater detail of mitochondrial structure. All nuclei exhibited red fluorescence, although the intensity varied considerably. Cells were then treated with either DMSO (B), 30 μM 4a (C), 100 μM dynasore (D) or 30 μM 6a. In (B) to (E), left-hand panels show images acquired 30 min after treatment, central panels show a more detailed image of mitochondria after 30 min of treatment and the right-hand panels show the cells after 60 min. After 30 min of treatment, 4a- and dynasore-treated cells exhibited unchanged mitochondrial morphology, including elongated mitochondria (arrows in A–D), while 6a-treated cells exhibited relatively fragmented mitochondria (arrows in E). After 60 min of treatment, all treated cells exhibited a reduction in Mitotracker Green FM fluorescence. Scale bars = 20 µm for images in left- and right-hand panels, while for zoomed panels the scale bar = 5 µm.
tra12119-sup-0009-FigureS6.docxWord 2007 document434KFigure S6. U2OS cells express only dynamin II. Equal protein load (50 µg) from four different cancer cell lines was run on SDS gels along with 0.2 µg partially purified full-length recombinant dynamin I, II or III. The three dynamins were detected with isoform-specific antibodies by western blot. Results shown are for one experiment with duplicate or triplicate cell samples and similar results were obtained in two additional experiments.
tra12119-sup-0010-FigureS7.docxWord 2007 document434KFigure S7. Dyngo compound 4a does not block dynamin-independent endocytosis of cholera toxin. A) NIH3T3 cells were serum starved for 3 h in unsupplemented DMEM. Cells were subsequently pretreated (or not) for 20 min with 20, 50 or 80 μM 4a or dynasore. Cells were next incubated with 5 µg/mL Tfn-488 and 2 µg/mL CT-555 in the continued presence of 20, 50 or 80 μM 4a or dynasore for 5 min at 37°C. 2 × 1-min washes with 0.5 M glycine and pH 2.2 were performed to remove surface labeling of CT-555 prior to fixation in 4% paraformaldehyde. Cells were imaged on a 510 Meta Zeiss confocal microscope. Scale bar is 10 µm. B) Over 50 cells treated with each condition in (A) were imaged and fluorescence intensity was calculated based on each unique histogram profile. Each treated sample was calculated as a percentage of control units. All data are means ± SEM.
tra12119-sup-0011-FigureS8.docxWord 2007 document659KFigure S8. Blockade of synaptic vesicle turnover in CGNs. A–D) Activity-dependent loading and unloading of the styryl dye FM1-43 in CGN cultures. Representative images either loaded at S1 (A) or S2 (B) in the absence of 4a or in its presence (S1—panel C; S2—panel D) are displayed. Scale bar represents 1 µm. E and F) Dextran endocytosis specifically reflects ADBE relative to CME. Representative images of control (E) or 4a (panel F, 30 μM, 15-min preincubation) inhibition of uptake of fluorescent tetramethyrhodamine-dextran (50 μM) in CGNs electrically stimulated by a train of 800 action potentials (80 Hz) followed by immediate dextran washout.
tra12119-sup-0012-FigureS9.docxWord 2007 document476KFigure S9. Dynasore inhibits SVE in neurons. The effect of dynasore on whole-cell membrane capacitance was investigated at the Calyx of Held in parallel with the experiments in Figure 4 except that there was 0.8 mM dynasore in the puffing pipette. A) A sample trace shows membrane capacitance on control (black) and dynasore-treated samples (red). B) Collated data of normalized capacitance measurement from control and dynasore-treated neurons (n = 7).

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