JCS03147 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. BFA treatment induces Golgi fragmentation in both SW13 v+ and v− cells. SW13 v+ and v− cells were either treated with methanol (Control) or 10 μg/ml BFA for 30 minutes at 37°C. Cells were then fixed and processed for immunofluorescence and stained with antibodies against the Golgi protein GM130. Confocal microscopy reveals that BFA induced Golgi fragmentation in both cell types. Scale bar, 10 μm.

• Supplemental Figure 2 -

  Fig. S2. Seeding of cells on the non-adherent substratum poly HEMA does not block BFA-induced changes in vimentin architecture. SW13 v+ cells were seeded on either Matrigel^TM or poly HEMA. Cells were then treated with either methanol (Control) or 10 μg/ml BFA for 30 minutes at 37°C. Cells were then fixed and process for immunofluorescence and stained with a monoclonal antibody against vimentin. Epifluorescence microscopy revealed that seeding of cells on the non-adhesive substratum matrix poly HEMA did not block BFA induced changes in the vimentin cytoskeleton. Scale bar, 20 μm.

• Supplemental Figure 3 -

  Fig. S3. BFA, but not IQ, results in the complete release of the adaptors AP-1 and AP-3 from the membrane (quantification of results from Fig. 4). We analyzed confocal images of AP-1 and AP-3 stained SW13 v+ cells treated in either the absence or presence of 10 μg/ml BFA or 40 μM IQ for 30 minutes at 37°C. Images were thresholded at an intensity set by Metamorph software to visualize membrane associated adaptor complexes. The combined area of all positive particles above the threshold was then calculated for each individual cell. The data are depicted as a range. The mean of condition is depicted as a horizontal line in the shaded gray box. The shaded box represents 50% of the data points, while the bars include 100% of the data points. BFA treatment resulted in a significant loss of membrane associated AP-1 and AP-3, whereas IQ treatment resulted only in minimal changes when compared to control untreated cells.

• Supplemental Figure 4 -

  Fig. S4. Inhibition of ADP ribosylation does not block BFA-induced changes in the vimentin cytoskeleton. (a) SW13 v+ cells were treated with either methanol or 10 μg/ml BFA for 30 minutes at 37°C either in the presence or absence of 50 mM nicotinamide, a general ADP ribosylation inhibitor. Cells were then fixed and processed for immunofluorescence and stained with antibodies directed against vimentin. Nicotinamide treatment alone caused minimal changes in vimentin networks. BFA-treated cells either in the presence or absence of nicotinamide exhibited process formation and retraction of the vimentin cytoskeleton, indicating that BFA induced changes were not due to induction of ADP ribosylation. (b) Nicotinamide inhibits nuclear ADP ribosylation. SW13 v+ cells were either untreated or treated with 1 mM hydrogen peroxide (H₂O₂) for 10 minutes at 37°C in order to induce nuclear poly ADP-ribosylation, Controls were performed either absence or presence of 50 mM nicotinamide or 20 units per ml catalase. Cells were processed and stained using antibodies against poly-ADP ribose. Epifluorescence microscopy revealed that hydrogen peroxide treatment induced nuclear poly-ADP ribosylation, which was not seen in either untreated or catalase-treated cells. Nicotinamide treatment blocked nuclear poly-ADP ribosylation, demonstrating that drug treatment was effective. Scale bars, 20 μm.

• Supplemental Figure 5 -

  Fig. S5. ARF1-T31N induces Golgi fragmentation. SW13 v+ cells were transiently transfected with GFP-tagged versions of either wild-type ARF1, ARF-T31N, or ARF-Q71L. Cells were fixed and processed for immunofluorescence and stained with antibodies directed against the Golgi protein GM130. Confocal imaging revealed that only the ARF1 mutant T31N, and not wild-type or Q71L, caused Golgi fragmentation. Scale bar, 10 μm.

• Movie 1 -

  Movie 1. BFA induces retraction and process formation arising from pre-existing vimentin filaments. SW13 v+ cells stably expressing GFP-vimentin were treated with 10 μg/ml BFA and imaged by spinning-disk confocal microscopy for 1 hour at 37°C. Images were taken every 45 seconds. The movie shows both retraction (upper left cell) and process formation (center cell) arising from pre-existing filaments.