Fig. 3

NWL-117 and −154 rapidly inhibit Casp6 activity in HCT116 cells. a Percent VEIDase activity from cellular protein extracts of Casp6-transfected HCT116 cells treated with either NWL-117 (closed triangle) or −154 (closed circle) at 100 μM for 0, 15, 30, 60, 90, or 120 min. No statistical significant differences were obtained between NWL-117 and NWL-154. b & c Western blot analysis of samples from panel (a) of NWL-117 (b) and NWL-154 (c) for α-tubulin-cleaved by Casp6 (TubΔCasp6), α-tubulin (Tubulin), active Casp6 p20 subunit (Casp6p20), and β-actin. d Percent VEIDase activity from cellular extracts after 2 h of treatment with 100 μM NWL-117 (closed triangle) or −154 (closed circle) in Casp6-transfected HCT116 cells followed by the removal of the inhibitors for 0, 15, 30, 60, 90, or 120 min. e & f Western blot analysis of samples from panel (d) of NWL-117 (e) and NWL-154 (f) for α-tubulin-cleaved by Casp6 (TubΔCasp6), α-tubulin (Tubulin), active Casp6 p20 subunit (Casp6p20), and β-actin. For panels (a) & (d), data represent the mean ± SEM of three independent experiments. Statistical analysis was performed by two-way ANOVA (((compound (p = 0.0010), time (p < 0.0001), interaction (p = 0.4546)); (compound (p < 0.0001), time (p < 0.0001), interaction (p = 0.0054))), respectively, with Bonferroni post-tests (*p < 0.05, **p < 0.01, ***p < 0.001)