Figure 5

Inhibitors of PMPMEase and GRK2i do not induce neuronal cell death. PC12 cells were grown on 96-well plates and treated with NGF for two days followed by incubation with 5 μM GRK2i for 10, 30, and 60 min (A). For PMPMEase inhibitors treatment, cells were seeded on 96-well plates and incubated simultaneously with NGF and PMSF, L-23, or L28 (5 and 10 μM) for two days (B). Subsequently, cells were incubated with a Hoechst/propidium iodide (PI) mixture for DNS cytotoxicity assay. The images were captured in live-cell-image mode using the confocal automated microscope BD Pathway Bioimager System and a 10× objective, assisted with AttoVision software. H₂O₂ (100 μM) was used as a positive control. Cell nuclei stained with Hoechst provided the total number of cells; cell nuclei stained with PI indicate the number of dead cells; merged Hoechst and PI images. Cell death was plotted as the percent of PI-positive cells, denoting the total number of dead cells for each condition.