Fig. 7

Endothelin A2 (Endo II) promotes the cytotoxicity of trastuzumab and T-DM1 in human epidermal growth factor receptor 2-positive (HER2+) cancer cells. a HCC1954 pLKO vector (Vec) and Endo II knock-down (KD)1 cells seeded in a 96-well plate were transfected with Nuclight2 live-cell nuclear stain, and treated with or without trastuzumab (TZ, 84 μg/ml) in medium supplemented with propidium iodide (PI; compared to control (ctl) with no drug added). Following real-time imaging, the relative increase in cytotoxicity (PI+ cells relative to Nuclight2+ cells) was analyzed over 48 hours for three experiments performed in triplicate. Representative merged images of red channel (PI) and bright field are shown on the left. The graph on the right indicate mean values (± SEM) from three experiments (significant differences between Vec and Endo II KD cells treated with TZ are denoted by *P < 0.05, **P < 0.01). b HCC1954 NT, Endo II KD1 and KD2 cells seeded in a 96-well plate were treated with or without trastuzumab emtansine (T-DM1) (50 ng/ml) in medium supplemented with PI compared to untreated control (Ctl). Following real-time imaging, the relative cell toxicity (calculated as PI+ cells relative to GFP+ cells) was analyzed over 48 hours for three experiments performed in triplicate. Representative merged images of red channel (PI) and bright field are shown on the left. The graph on the right indicates mean values (± SEM) from three experiments (significant differences between non-targeted (NT) and Endo II KD cells treated with T-DM1 are denoted by ***P < 0.001)