Additional file 8 of Histamine N-methyltransferase (HNMT) as a potential auxiliary biomarker for predicting adaptability to anti-HER2 drug treatment in breast cancer patients

Additional file 8: Fig. S8 Receptor-mediated interactions between HER2 and HNMT in BC cells. (A) BC cells were treated with EGF (100 ng/mL) in a time-dependent manner (0, 10, 20,30, and 60 min), and immunoblot analysis revealed HNMT protein expression in subcellular fractions. (B) Proteins isolated from EGF-treated SKBR3 cells in a time-dependent manner (0, 20, and 50 min), and the cells were immunoprecipitated to observe the interaction between the HNMT, HER1 and HER2 proteins. (C) Schematic representation of the ligand-mediated interaction between the HNMT and HER2 proteins at the cell membrane (left) at 20 min. Representative images and quantitative results of FRET efficiency in SKBR3 cells treated with and without EGF or NRG (100 ng/mL and 100 μM, respectively). Yellow arrows indicate a positive FRET signal. The data are presented as the means ± SE. Statistical analysis was performed using a two-tailed unpaired Student's t-test. ***P < 0.001. (D) The Supplementary Data of Fig. 3A shows schematic representations of molecular interactions and IF images with positive (right) or negative (left) FRET signals. (E) After treating SKBR3 cells with EGF (100 ng/mL) for 20 min, the FRET technique was employed to analyze the potential interaction between HER1 and HNMT. (F) SKBR3 cells were transiently transfected with CRISPR control and HER1 KO plasmids. Western blot analysis confirmed the expression of the indicated proteins in SKBR3 stable cells (SK-Vc and SK-HN). After the cells were treated with and without EGF (100 ng/mL) for 20 min, the interaction between HNMT and HER2 protein on the cell membrane was observed. It shows that the interaction between HNMT/HER2 is obvious in the presence of EGF (red arrow). Without HER1 protein, the interaction between HNMT and HER2 protein is blocked. Scale bar=10 µm (C, D, E, F).