The Journal of Neuroscience, July 12, 2006, ():
Pancreatitis-Associated Protein-III Is a Novel Macrophage Chemoattractant Implicated in Nerve Regeneration
J. Neurosci. Namikawa et al.
26: 7460
Supplemental data
Files in this Data Supplement:
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Supplemental methods
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Supplemental Figure 1. (A) RT-PCR demonstrating remarkable expression of PAP-III mRNA in the distal parts of injured sciatic nerves at 1 day after axotomy. MCP-1 mRNA expression is also rapidly induced in the injured nerves. The expression of GAPDH mRNA was used as an internal control. (B) SDS-PAGE analysis and subsequent silver staining showing that the purified recombinant PAP-III is exactly the same size as the secreted PAP III protein (arrow) in the supernatant (sup.) of 293T cells transfected with a PAP-III-expressing plasmid. These bands are also recognized by western blotting analysis using an anti-PAP-III antibody (lower).
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Supplemental Figure 2. Effect of a siRNA expression vector against PAP-III. Different amounts of plasmids expressing PAP-I or PAP-III were co-transfected into COS7 cells together with 300 ng of a siPAP-III-expressing plasmid. Subsequently, western blotting analyses were performed on the cell lysates. Although siPAP-III expression has no effect on the expression of PAP-I (A), it effectively reduces PAP-III expression in a dose-dependent manner (B). The same membrane was reprobed with an anti-GAPDH antibody to confirm that equal amounts of protein were loaded.
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Supplemental Figure 3. PAP-III has no activity as a Schwann cell mitogen in vitro. (A) A Schwann cell marker, S100 (green) and an indicator of DNA synthesis BrdU (red) double staining of Schwann cells in the absence (left) or presence of PAP-III (middle) or ß-Heregulin (right), one of well-known Schwann mitogens, at the concentration of 50ng/m, respectively. Forskolin (10 µM) was added to the cultures together with these factors. (B) The mean percentage of Schwann cells (S100 positive cells) double-labelled for BrdU incorporation in the 24 h following each treatment. Note that PAP-III failed to stimulate Schwann cell growth even in the presence of Forskolin, whereas the growth of the cells stimulated by ß-Heregulin (Her) was significantly enhanced especially in the presence of Forskolin. Error bars indicate SEM. Scale bar represents 50 µm (A).
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Supplemental Figure 4. PAP-III has no effect on neurite outgrowth in DRG organ cultures. (A) DRGs were embedded in the collagen gels and cultured for 4 days in the absence (upper; cont) or presence of PAP-III (middle) or NGF (lower) at the concentration of 50ng/ml, respectively. Their neurites were visualized with anti-neurofilament antibody. (B) Neurite length was measured from the surface of DRG. Application of PAP-III at various concentrations has no impact on enhancment of the neurite outgrowth of DRG cells, compared to NGF. Error bars represent SEM. Scale bar represents 500 μm (A)
