Afatinib

Cells were seeded in 10 cm dishes 1 day before treatment. MKN1, MKN7 and Hs746T cells were plated at a density of 1.7 × 10⁴ cells/cm² and NCI-N87 at 2 × 10⁴ cells/cm². Medium was changed 2 h before treatment. Cells were treated with EGF (5 ng/ml, Sigma Aldrich), cetuximab (Cet, 1 μg/ml, Merck), trastuzumab (Tra, 5 μg/ml, Roche), afatinib (Afa, 0.5 μM, Biozol) or dimethylsulfoxid (DMSO, 0.05%, afatinib solvent control) for 4 h or 24 h. RNA and micro RNA were isolated using the mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific), according to manufacturer’s instructions. The RNA was eluted in nuclease-free water. DNase digestion was performed using the DNA-free™ DNA Removal Kit (Thermo Fisher Scientific) according to manufacturer’s instructions.

Cells were seeded in 10 cm dishes one day before treatment (cell numbers see Table S1, Additional file 1) and subsequently treated with EGF (5 ng/ml, Sigma Aldrich), cetuximab (Cet, 1 µg/ml, Merck), trastuzumab (Tra, 5 µg/ml, Roche), afatinib (Afa, 0.5 µM, Biozol) or dimethylsulfoxid (DMSO, 0.05%, afatinib solvent control) for 4 h or 24 h. RNA and micro RNA were isolated using the mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific) and RNA was eluted in nuclease-free water. The DNA-free™ DNA Removal Kit (Thermo Fisher Scientific) was used for DNase digestion according to manufacturer’s instructions. All experiments were performed in triplicate.
The treatment times of 4 h and 24 h were chosen because of literature, previous experiments and duration of phenotypic analyses. The 4 h treatment was chosen because it corresponds to the middle of the film length of 7 h. The 24 h treatment was chosen since apoptosis was analyzed 24 h after treatment and effects on gene expression were shown in breast cancer cell lines after 24 h trastuzumab treatment [20 (link)]. Moreover, this time was chosen since previous gene expression experiments with cetuximab were performed after 24 h treatment.

Using a Luminex® xMAP® platform in a magnetic bead format, we simultaneously studied the following analytes from the culture supernatants of MUG-Chor1 and UM-Chor1 cells: epidermal growth factor (EGF), tumour necrosis factor alpha (TNFα), vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2). No cross-reactivity was noted between the antibodies for an analyte or between any of the other analytes in this panel. Treatment was performed with 4 μM crizotinib (PF-02341066; SelleckChem, Houston, TX, USA), 0.16 μM afatinib (BIBW2992; SelleckChem), or with a simultaneous co-dosing of crizotinib and afatinib for 72 h. For detection, we used a commercially available Procarta Plex (Thermo Fisher, Waltham, MA, USA) on a Bioplex200 system (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturers´ instructions. Measurement of mean fluorescence intensities was performed using Bio-Plex Manager software, version 4.1 (Bio-Rad Laboratories, Hercules, CA, USA) with a 5-parametric curve fitting.