Fig. 4
Non-toxic concentrations of NWL-117 and −154 inhibit Casp6 activity in primary human neurons. a Micrographs of primary human neurons treated with PBS vehicle, 100 μM NWL inhibitors, or 2 μM staurosporine for 24 h. Scale bar represents 10 μm. b MTT absorbance following treatment with PBS, NWL-117, or NWL-154 at 20, 50, or 100 μM or 2 μM staurosporine for 24 h (n = 4, one-way ANOVA (p = 0.0169), Tukey’s multiple comparison test (*p < 0.05)). c VEIDase activity in neuronal extracts following treatment with PBS, 100 μM NWL-117 (n = 5), or 154 (n = 2) for 2 h (one-way ANOVA (p < 0.0001)). d Casp6 FLICA assay (one-way ANOVA (p = 0.0041)). e Quantification of the number of TubΔCasp6 beads/nuclei in Additional file 1: Figure S2b (one-way ANOVA (p = 0.0095)). f–h Fluorescence micrographs following transfections with pBudEGFP (f) or pBudEGFP/APPWT (g) stained for α-tubulin (Cy3), Hoechst, and quantified (h) (one-way ANOVA (p = 0.0385)). Scale bar represents 100 μm for merge and Hoechst panels, and 50 μm for EGFP panel. i Live-imaging fluorescence micrographs from 0 to 60 h of a human neuron transfected with pBudEGFP/APPWT and pre-treated with vehicle. The inset highlights the neurite extending upward. Arrowheads indicate agglomerates of EGFP protein within the axonal membrane while arrows mark a rounded cell body. Scale bar represents 10 μm. j–m Quantification of panel (i) for overall fold increased beaded neurites (j), fold increased beaded neurons at specific times (k), overall fold increase swollen neuronal soma (l), or normal EGFP positive neurons (m). For panels (c–e, h), and (j–k) data represent the mean ± SEM (n ≥ 3), one-way ANOVA (p = 0.0005 for j, p =0.0264 for k, and post hoc tests were performed with Dunnett’s multiple comparison test (*compares to serum (+) or EGFP-vehicle: *p < 0.05, **p < 0.01, ***p < 0.001; # compares to serum (−) with vehicle or to EGFP/APPWT-vehicle: # p < 0.05, ## p < 0.01, ### p < 0.001) unless stated otherwise. For panel (m), log-rank Mantel-Cox test was performed to compare between curves