Fig. 5

Simultaneous inhibition of human epidermal growth factor recptor-2 (HER2), p110α and Vps34 induces cell death in HER2+ breast cancer. a UACC893, BT474 and SKBR3 cells were cultured with 1 μM SAR405, 0.5 μM Lapatinib or 0.5 μM BYL719. Relative cell number was determined by sulforhodamine B assay at time 0 and after three days of culture. Day 3 signal divided by day 0 signal was used to normalize cell number to day 0. b Cells were treated as described in a. Lysates were harvested 6 h after treatment and analyzed by immunoblot analysis with the indicated antibodies. c UACC893-GFP-LC3 cells were treated with 1 μM SAR405, 0.5 μM Lapatinib or 0.5 μM BYL719 for 3 h before being fixed and green fluorescent protein (GFP)-LC3 localization determined by fluorescent microscopy. SKBR3-GFP-LC3 cells were analyzed similarly, but fixed after 6 h of treatment. d, e UACC893 and BT474 cells were treated for 6 h and SKBR3 cells for treated 24 h with 1 μM SAR405, 0.5 μM Lapatinib or 0.5 μM BYL719 before caspase 3/7 activity was determined (d) or stained with Annexin V (e). In all experiments, SAR405 was applied 30 minutes prior to the application of BYL719, lapatinib or dimethyl sulfoxide (DMSO) control (*p <0.05, Student t test comparing the effect of DMSO vs. SAR405 within each treatment group)