Fig. 2
Non-toxic concentrations of NWL-117 and −154 inhibit Casp6 activity in HCT116 cells. a Phase contrast microscope images of HCT116 cells treated with phosphate-buffered saline, 100 μM NWL inhibitors, or 2 μM staurosporine for 24 h. Scale bar represents 10 μm. b & c MTT absorbance following treatment with PBS, NWL-117, or NWL-154 at 20, 50, or 100 μM for 24 h (b) or 48 h (c). No statistical differences were found by two-way ANOVA with Bonferroni post-tests. d Lactate dehydrogenase activity in untreated cells, or treated with PBS, 100 μM NWL inhibitors, or lysed with 0.9% Triton X-100 for 24 h. e Quantification of the sub-G1 population following cell cycle analysis in PBS, 100 μM NWL inhibitors, or 2 μM staurosporine treatment for 24 h. f VEIDase activity in pCep4β-transfected (mock) or pCep4β-Casp6p20p10 transfected HCT116 cells. Data represent the mean ± SEM of five independent experiments. Statistical analysis was performed using an unpaired two-tailed t-test (*** p < 0.01). g Casp6 VEIDase activity in cellular extracts from pCep4β-Casp6p20p10-transfected HCT116 cells treated with PBS, NWL-117 or NWL-154 at 100 μM for 2 h. h Dose-response curve for NWL-117 (closed circle, IC50 = 4.82 μM) and NWL-154 (open circle, IC50 = 3.63 μM) in pCep4β-Casp6p20p10-transfected HCT116 cells treated for 2 h. i Western blot analysis of samples from panel f and g for α-tubulin-cleaved by Casp6 (TubΔCasp6), α-tubulin (Tubulin), active Casp6 p20 subunit (Casp6p20), and β-actin. Casp6 expression was allowed for 24 h before treatment with inhibitors in all transfection experiments. For panels b-g, data represent the mean of three independent experiments ± SEM and were analyzed by one-way ANOVA (p < 0.0001) with post hoc Dunnett’s multiple comparison test comparing to vehicle-treated (***denotes p < 0.001) unless specified otherwise