Additional file 10 of Histamine N-methyltransferase (HNMT) as a potential auxiliary biomarker for predicting adaptability to anti-HER2 drug treatment in breast cancer patients

Additional file 10: Fig. S10 HNMT expression affects the binding of HER2+ tumor cells to trastuzumab. (A) The cellular localization of different parts of the HER2 protein was confirmed by IF staining. Schematic representation of regions recognized by trastuzumab and anti-HER2-ICD antibodies (left). Representative images and qanitification data of IF double-stained cells showing the cellular distribution of HNMT and HER2 isoforms by 100 ng/mL EGF treating 20 min. The right panel shows HER2-ICD detection using the HER2-ICD antibody (green) and p185HER2 detection using trastuzumab (green). The white arrows indicate the nuclear localization of HER2-ICD, while the yellow arrows indicate negative results. n=5 biologically independent experiments. Scale bar = 10 µm. (B) The frequencies of the top 50 ranked target genes were determined by ChIP-sequencing analysis. (C) pGL3-4X-HBS-1 and pGL3-4X-HBS-2 were transfected into SKBR3 cells expressing the HER2 scramble or HER2 siRNA, respectively. Quantitative luc reporter assays were performed following the treatment of transfected cells with/without EGF or NRG1. The pGL3 vector was transfected into SKBR3 cells as a control. The pRL-TK plasmid was used as an internal control. (D) Western blotting confirmed HNMT and HER2 protein expression in the HER2+ BC cell lines harboring vectors overexpressing HNMT, HNMT scrambled RNA or HNMT siRNA. (E) The ability of trastuzumab to bind to the HER2 protein in the above four cell lines (D) was detected by flow cytometry. n = 3 biologically independent experiments. The data are presented as the mean ± SE. Statistical analysis was performed using a two-tailed unpaired Student's t-test. *P < 0.05, **P < 0.01, and ***P < 0.001.