Supplemental Material

This article contains the following supporting material:

• Figure 1 - Specificity of staining

  Control for the experiment shown in Fig. 2A-C. The first antibody against VCP has been omitted. (A) DAPI stain shows location of nuclei. (B) Wld^S/EGFP signal localizes largely to intranuclear spots (arrowhead). (C) In the absence of first antibody there is no red signal. (D) Overlay.

• Figure 2 - Wld^S partially redistributes VCP in HeLa cells

  As in PC12 cells, Wld^S/EGFP localizes to intranuclear spots and VCP colocalizes with it (arrowhead), while retaining its more general distribution in nucleus and cytoplasm. (A) DAPI stain shows location of nuclei. (B) Wld^S/EGFP signal localizes to intranuclear foci. (C) VCP immunostain (D) Overlay.

• Figure 3 - Expression of Wld^S/EGFP and consequent redistribution of VCP is harmless in PC12 cells

  Consistent with the fact that Wld^S is harmless in vivo (Lunn et al., 1989; Mack et al., 2001; Adalbert et al., 2005) and in vitro (Araki et al., 2004), PC12 cells expressing Wld^S/EGFP display a healthy, flattened appearance with a centrally located nucleus of normal size, and even begin to extend neurites (arrow in D). (A) DAPI stain shows location of nucleus. (B) Wld^S/EGFP signal localizes to intranuclear foci (arrowhead). (C) VCP immunostain with similar pattern of intranuclear foci (D) DIC image (* marks a little extracellular debris which lies over the upper part of the cell) (E) Overlay shows that VCP signal was detected throughout the cell, apart from some peripheral regions that lie outside the confocal slice.

• Figure 4 - Expression of Wld^S/EGFP and consequent redistribution of VCP is harmless in HeLa cells

  Consistent with the fact that Wld^S is harmless in vivo (Lunn et al., 1989; Mack et al., 2001; Adalbert et al., 2005) and in vitro (Araki et al., 2004), HeLa cells expressing Wld^S/EGFP mostly display a healthy, flattened appearance with a centrally located nucleus of normal size. (A) DAPI stain shows location of nuclei. (B) Wld^S/EGFP signal localizes to intranuclear foci (arrowhead). (C) VCP immunostain, showing intranuclear foci in transfected cells (D) DIC image (E) Overlay shows that VCP signal was detected throughout both transfected and untransfected cells, apart from some peripheral regions that lay outside the confocal slice.

• Figure 5 - Wld^S and VCP colocalize in DRG and in Wld^S rats

  (A, B, C) Partial colocalization in intranuclear foci (arrowhead) in Wld^S mouse DRG suggests that Wld^S and VCP interact in neurons that show the slow Wallerian degeneration phenotype. Scale bar: 5 μm. (D, E, F) Colocalization in a rat DRG neuron indicates that this interaction is conserved between mammalian species. Scale bar: 5 μm (G, H, I) Motor neurons in vitro, electroporated with Wld^S/EGFP sometimes show Wld^S puncta (green; G), unlike their in vivo counterparts (Mack et al., 2001). VCP immunocytochemistry (red; H) shows a punctate distribution, but the spots are smaller and do not specifically colocalize with Wld^S/EGFP (note: green and red are reversed with respect to A-E) (I). Neurofilament medium chain staining (blue) confirms the neuronal identity, scale bar: 10 μm. Each figure is representative of two or more experiments.

• Figure 6 - TV cells express endogenous VCP throughout the cell

  As previously reported (Kobayashi, et al., 2002), the PC12 subline TV displays a normal distribution of endogenous VCP throughout the cytoplasm and nucleus (B). The figure shows cells grown in the presence of doxycycline, so that endogenous VCP can be distinguished from ectopic VCP/EGFP. (A) Control lacking first antibody demonstrates specificity of VCP staining. (B) VCP immunostain. (C, D) DAPI staining shows location of nuclei. (E, F) phase contrast image showing that the majority of cells have a flattened, healthy appearance.