Pep2m

Myristoylated ZIP, pep2m, and scrambled peptide were acquired from Tocris Bioscience; prostaglandin E₂ (PGE₂) was obtained from Cayman Chemical Company; and nerve growth factor (NGF) was from R&D Systems. ZIP, pep2m and scrambled peptide stock solutions were prepared in sterile 0.9% saline and diluted from this stock solution for injection. PGE₂ stock solutions were prepared in 100% ethanol and diluted in sterile saline for injection. Doses used in this study were based on our previous work (Asiedu et al., 2011 ) and other publications (Shi et al., 2001 (link), Pastalkova et al., 2006 (link), Shema et al., 2007 (link)). ZIP is not a selective inhibitor of aPKCs (Lee et al., 2013 (link), Volk et al., 2013 (link)). pep2m interferes with GluA2 binding to NSF and the effect of the peptide is absent in GluA2 KO mice, suggesting it is a specific tool peptide (Shi et al., 2001 (link)).

For migration assays, a total of 1x10⁵ Ishikawa cells in 200μl of serum-free DMEM medium was seeded into the upper chamber of a 24-well polycarbonate transwell filter (8μm pore, Corning Inc., Glendale, AZ, USA), and 600μl of complete medium was added to the lower chamber. The cells were treated with GluR2 antagonists (10μm, PEP2M, TOCRIS, UK) and agonist (10μm, CI-HIBO, TOCRIS, UK). The cells were fixed with 4% paraformaldehyde, stained with crystal violet staining solution (Beyotime, Shanghai, China) and counted at 200x magnification in five random fields/well. The invasion of cells was performed using transwell chambers with 8μm pore membranes precoated with 50 μl of Matrigel at 1:6 dilution (BD Biosciences, San Jose CA, USA) on the upper side at 37°C for 1 h. The following process was the same as described above. As in the indirect coculture system using transwells, Ishikawa cells were seeded in the upper chamber, and DRG neuron cells were seeded in the lower chamber. Migrated cells were counted as previously described. In the meantime, the condition medium after coculture was collected and used in subsequent experiments.