Inhibitors of PMPMEase disrupt neurite outgrowth of NGF-differentiated PC12 cells. PC12 cells were treated with PMPMEase inhibitors, L-23 and L-28 (5 μM, and 10 μM), or the prototypical molecule PMSF (10 μM) and allowed to differentiate in the presence of 100 ng/mL of NGF for two consecutive days. (A) The cells were then fixed and double labeled with anti-tubulin (red) and anti-Gβ (green) antibodies, and DAPI was used for nuclear staining (blue). Co-localization patterns are also shown in the merged images. PMSF did not seem to have any significant effects on neuronal morphology (a–d). PMPMEase inhibitors inhibited neurite outgrowth of NGF-treated PC12 cells, causing axonal damage (e–h, enlarged image shown in h’), neurite shortening (i–l, enlarged image shown in l’), and cellular aggregation (m–p). Scale bars are 20 μm (B–C). Using Zeiss ZEN software, neurites were traced and measured, and the average neurite length and percent of cells bearing neurites were estimated. The differences between experimental conditions were assessed by one-way ANOVA. *p < 0.05; **p < 0.01.