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Trastuzumab causes cell cycle arrest and cell death in trastuzumab-hypersensitive cells. Trastuzumab-resistant and trastuzumab-hypersensitive models maintain HER2 levels ( A - E ). HER2 surface levels were analyzed by flow cytometry ( A ), immunofluorescence ( B ) and cell surface immunoprecipitation ( E ) using trastuzumab 10 nM as primary antibody in all cases. Total HER2 levels were determined by immunofluorescence ( C ) or western blot ( D ). Levels of total ( D ) and cell surface tyrosine-phosphorylated HER2 were determined by western blot. For immunofluorescence, cells were fixed and stained for HER2 using trastuzumab (green) ( B ) or an anti-HER2 antibody (red) ( C ) and DNA (blue). Scale bar, 10 μm. F Evaluation of S phase entry upon trastuzumab treatment. Cells were treated with trastuzumab for 5 days, received a 10 μM pulse of BrdU and BrdU incorporation was determined by flow cytometry. Data is represented as the mean ± SD, normalized to untreated controls of two independent experiments. G Effect of trastuzumab on cell death. Cells were treated with trastuzumab for 6 days and stained with annexin V-FITC and propidium iodide, followed by flow cytometry analysis. The graph shows mean ± SD of viable and non-viable cells from two independent experiments. I Trastuzumab treatment induces caspase 3 activation in BTSH cells. Cells were treated with trastuzumab for 6 days and caspase 3 activity was determined by fluorometric techniques using a caspase 3 specific substrate. H Analysis of proapoptotic and cell cycle progression proteins after trastuzumab treatment. Cells were treated with trastuzumab for 6 days and the amount of the indicated proteins was assessed by western blot. cCaspase stands for cleavage caspase. Calnexin was used as a loading control. C: control; T: trastuzumab; IP: immunoprecipitation; WB: western blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Modelling hypersensitivity to trastuzumab defines biomarkers of response in HER2 positive breast cancer

doi: 10.1186/s13046-021-02098-z

Figure Lengend Snippet: Trastuzumab causes cell cycle arrest and cell death in trastuzumab-hypersensitive cells. Trastuzumab-resistant and trastuzumab-hypersensitive models maintain HER2 levels ( A - E ). HER2 surface levels were analyzed by flow cytometry ( A ), immunofluorescence ( B ) and cell surface immunoprecipitation ( E ) using trastuzumab 10 nM as primary antibody in all cases. Total HER2 levels were determined by immunofluorescence ( C ) or western blot ( D ). Levels of total ( D ) and cell surface tyrosine-phosphorylated HER2 were determined by western blot. For immunofluorescence, cells were fixed and stained for HER2 using trastuzumab (green) ( B ) or an anti-HER2 antibody (red) ( C ) and DNA (blue). Scale bar, 10 μm. F Evaluation of S phase entry upon trastuzumab treatment. Cells were treated with trastuzumab for 5 days, received a 10 μM pulse of BrdU and BrdU incorporation was determined by flow cytometry. Data is represented as the mean ± SD, normalized to untreated controls of two independent experiments. G Effect of trastuzumab on cell death. Cells were treated with trastuzumab for 6 days and stained with annexin V-FITC and propidium iodide, followed by flow cytometry analysis. The graph shows mean ± SD of viable and non-viable cells from two independent experiments. I Trastuzumab treatment induces caspase 3 activation in BTSH cells. Cells were treated with trastuzumab for 6 days and caspase 3 activity was determined by fluorometric techniques using a caspase 3 specific substrate. H Analysis of proapoptotic and cell cycle progression proteins after trastuzumab treatment. Cells were treated with trastuzumab for 6 days and the amount of the indicated proteins was assessed by western blot. cCaspase stands for cleavage caspase. Calnexin was used as a loading control. C: control; T: trastuzumab; IP: immunoprecipitation; WB: western blot

Article Snippet: Caspase 3 activity after 6 days trastuzumab 50 nM treatment was fluorometrically determined by the caspase 3 activity reporter Ac-DEVD-AFC (BD Biosciences, Palo Alto, USA) as described [ ].

Techniques: Flow Cytometry, Immunofluorescence, Immunoprecipitation, Western Blot, Staining, BrdU Incorporation Assay, Activation Assay, Activity Assay