JCS02358 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. tGolgin-1 depletion does not affect the distribution of endosomal markers. HeLa cells transduced with siRNA-1 were fixed and labelled with antibodies to TGN46 (a,d,g) and to the early endosome marker EEA1 (b) or the late endosome/lysosome markers CD63 (e) or Lamp1 (h), followed by fluorochrome-conjugated secondary antibodies. Cells were analysed by fluorescence microscopy. Deconvolved images of a single plane of focus are shown. (c,f,i) Overlaid images from both labels. Notice that the pattern of staining for the endosomal markers in cells with dispersed TGN46 labelling is indistinguishable from that in cells with a tight paranuclear TGN46 label.

• Supplemental Figure 2 -

  Fig. S2. VSV-G/EGFP transport in control cells and in cells overproducing dynamitin-p50. HeLa cells were transfected with plasmid encoding VSV-G/EGFP and either a control plasmid or dynamitin-p50, as indicated. Cells were grown at 39°C overnight and then fixed either immediately (at 39°C) or after 30 minutes or 60 minutes at 32°C, as indicated. Cells were analysed by fluorescence microscopy after labelling with anti-TGN46 and RRX-conjugated anti-sheep IgG. Deconvolved images are shown of individual z-axis sections.

• Supplemental Figure 3 -

  Fig. S3. Accumulation of STxB in transferrin-receptor- and EEA1-containing endosomes in cells depleted of tGolgin-1. HeLa cells transduced twice with control siRNA (a-d) or siRNA-1 (e-h) were incubated with Alexa-488-conjugated STxB on ice for 45 minutes, washed and then incubated at 37°C for 60 minutes. Cells were then fixed and labelled with antibodies to TGN46 and to transferrin receptor (TfR) (A) or EEA1 (B), followed by AMCA-labelled anti-sheep IgG and RRX-labelled anti-mouse IgG. Cells were analysed by three-colour fluorescence microscopy for STxB (Aa,e; Ba,e), TfR (Ab,Af) or EEA1(Bb,Bf) and TGN46 (Ad,h; Bd,h), the last of these to identify phenotypic tGolgin-1-depleted cells. (Ac,g; Bc,g) Overlay of the STxB and TfR (A) or EEA1 (B) signals; insets, twofold magnified images of the boxed regions.