Fig. 5
ITCH silencing or chemical inhibition blocks 9F7-F11-induced apoptosis and c-FLIP degradation. siSC- and siITCH-transfected BxPC3 cells were incubated with 9F7-F11 alone (a), or with NRG1 (b) for 48 h before detection by western blotting of ITCH and c-FLIPL expression, and PARP/caspase cleavage. MDA-MB-468 cells were incubated with increasing doses of chlorimipramine (CI) for 24 h (c), or with 15 μM CI (d) before incubation with 9F7-F11 for 24 h. Total protein extracts were analyzed by western blotting to evaluate PARP and caspase cleavage (c), ITCH ubiquitination and expression and c-FLIP and HER3 expression (d). Quantification of signal intensity (SI) with ImageJ software is indicated below the images. No protein expression was measured as 0.0 ± .0. Significant decrease or increase of the densitometry, compared to control, is indicated in bold. BxPC3 cells were left untreated or pre-incubated with ITCH chemical inhibitor CI for 48 h before treatment with 9F7-F11 with or without NRG1. Apoptosis was measured at 96 h by flow cytometry after cell labelling with Annexin V/7-AAD (e). siSC and siITCH-transfected BxPC3 cells were treated with 9F7-F11 alone or with NRG1. Western blot was performed to check ITCH reduction in siITCH-transfected BxPC3 cells. Apoptosis was measured at 96 h by flow cytometry after cell labelling with Annexin V/7-AAD (f). **P < 0.01, ***P < 0.001, ns not significant