JCS010769 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. HeLa cells were untreated (A), treated with 5 μg/ml BFA for 30 minutes (B) or transfected with vector encoding the E794K mutant of GBF1 for 24 hours (C), and stained with antibodies against the indicated proteins. BFA and GBF1-E794K cause dissociation of β-COP and the collapse of the Golgi. Bars, 16 μm.

• Supplemental Figure S2 -

  Fig. S2. Control HeLa cells or cells transfected with siRNA against GBF1 for 72 hours were stained with the indicated antibodies. The Golgi-derived tubules align on microtubules (A). The architecture of the microtubule cytoskeleton is not disrupted by depletion of GBF1. Bars, 16 μm.

• Supplemental Figure S3 -

  Fig. S3. Depletion of GBF1 inhibits cell migration. D54 human carcinoma cells were left untreated (A; control), transfected with scrambled siRNA (B; ‘scramble’) or with siRNA against GBF1 (C; ‘siRNA’). After 72 hours, cells were plated on top of trans-well filters and their migration to the bottom of the filters assayed 6 hours later. Representative images of the bottom of the trans-well filters are shown. Quantitation of 15 random fields for each condition is shown in D. (E) To indicate depletion of GBF1 cell lysates were analysed by western blotting with anti-GBF1 and anti-β-tubulin antibodies. β-tubulin provided the loading control.

• Movie S1 -

  Movie S1. NRK cells 72 hours after transfection with siRNA against GBF1 were processed for immunofluorescence with an antibody against GM130. A stack of 20 images was taken from the cells expressing the GBF1-depleted phenotype. Image acquisition was with the 100× oil 1.4 NA objective of a Leica DMIRBE inverted epifluorescence microscope outfitted with Leica TCS NT laser confocal optics (Leica Microsystems, Bannockburn, IL). A 488-nm high-intensity argon laser beam was set up at high intensity with a laser set on one-directional scan at speed ‘slow 2’. Each image round of scanning took 3 seconds in the 1024×1024 format. Every image was an average of four frames. Optical sections through the Z axis were generated using a computer-controlled focus step motor. Flattened projection of image stacks and a 3D rendering were prepared using proprietary confocal imaging software from Leica. A composite image is presented in Figure 2A.

• Movie S2 -

  Movie S2. HeLa cells 48 hours after transfection with siRNA against GBF1 were co-transfected with vectors encoding GRASP65-GFP and ERGIC58-YFP. After an additional 24 hours of incubation, cells were imaged with a Leica DMRXE upright, epifluorescence-Nomarski microscope outfitted with Leica TCS SP2 laser confocal optics. The system was equipped with argon ion, solid-state and helium-neon lasers for the imaging of a wide range of green and yellow fluorochromes. Precise control of fluorochrome excitation and emission is afforded, respectively, by an acousto-optical tunable filter and a TCS SP2 prism spectrophotometer. The objective used in the experiment was 100× oil. Exact settings to distinguish YFP and GFP were: GFP settings: line 488-100%, PMT1, band-pass 496-515, pinhole 370, dichroic−RSP 500, rheostat−3 o’clock; YFP settings: line 514-100%, PMT 2, band-pass 565-612, pinhole 370, dichroic- DD 458/514, rheostat−3 o’clock. Two color images were obtained every 3 seconds during the time of image acquisition. Sample images from this video are presented in Figure 2D−G. For ease of visualization, the yellow in these figures was digitally changed to red after image acquisition.