Atglistatin

Red C12 experimental methods were performed similar to what was described previously (Rambold et al., 2015 (link)). Atglistatin (Sigma) was added, where indicated, at 10uM concentration for the duration of assay. Images were taken using the Zeiss 710 Confocal Microscope.

PEDF and its derived peptides, 17-mer, 44-mer, and 34-mer were diluted in HBSS and aliquots were added to the cultures at 10 nM final concentration 48 hr after seeding the cells. The 17-mer and 44-mer peptides from the neurotrophic domain were the effectors and the 34-mer peptide from the antiangiogenic domain of PEDF was used as a negative control. For suppressing the effects of PEDF and its derived peptides, a fragment of the ligand-binding site of PEDF-R, the P1 blocking peptide, diluted in HBSS was added at a P1-PEDF molar ratio of 10:1. Alternatively, a selective inhibitor of PEDF-R, Atglistatin (Sigma, SML1075) at a 3.5 μM concentration (aliquoted and diluted in DMSO) was used. Both blocking peptide and inhibitor were preincubated in the cultures for 1 hr before adding the effectors. Preliminary experiments with 10 nM and 100 nM PEDF showed that 10 nM PEDF was the optimum PEDF concentration to promote survival of photoreceptors. The reported affinity of the PEDF/PEDF-R interactions is within this value (Kd of ~3 –10 nM PEDF) and the concentration of 10 nM PEDF has been used in assays with other cells such as R28 cells, which efficacy is blocked with 10-fold molar excess of P1 peptide or in the presence of 3.5 μM Atglistatin (Kenealey et al., 2015 (link); Notari et al., 2006 (link); Subramanian et al., 2013 (link)).