Fig. 6

FTI treatment increases LAMP1 in proximal and distal neurites in mouse primary neurons. Wild-type primary forebrain neurons were grown for seven days in culture and treated for 48 hours with vehicle (DMSO), 10 nM LNK-754 or 10 nM lonafarnib. A-C Confocal immunofluorescence images showing cells immunostained for LAMP1 (green) and β3-tubulin (blue). Magnified images show LAMP1 in neurites. Scale bars, 50 μm, 10 μm (magnified images). Quantification of LAMP1-covered area in cell neurites (**p = 0.0061) (D) soma (E) and total area (**p = 0.0045) (F). N = 3 independent experiments, values from individual cells are plotted. 10–16 cells were analyzed from 2 to 3 coverglasses per treatment group in each experiment. Total number of cells analyzed: vehicle, 44; LNK-754, 43; lonafarnib, 43. G Immunoblot of cell lysates from vehicle, LNK-754 and lonafarnib treated wild-type primary neurons probed for LAMP1 and actin. H Quantification LAMP1 immunoblot in (G). Signals were normalized to actin and expressed as fold of vehicle (*p = 0.013 between vehicle and LNK-754, ***p = 0.0008 between vehicle and lonafarnib). I Confocal immunofluorescence images showing cells stained with LysoSensor-Green. Scale bars, 25 μm. Arrows denote LysoSensor vesicles in distal neurites. J Quantification of LysoSensor-Green fluorescence intensity. N = 3 independent experiments, values from individual cells are plotted. 10–16 cells were analyzed from 2 to 3 coverglasses per treatment group in each experiment. Total number of cells analyzed: vehicle, 39; LNK-754, 36; lonafarnib, 38. 1-way ANOVA with Tukey’s post-hoc multiple comparisons test was performed