Additional file 3 of Histamine N-methyltransferase (HNMT) as a potential auxiliary biomarker for predicting adaptability to anti-HER2 drug treatment in breast cancer patients

Additional file 3: Fig. S3 In vitro and in vivo carcinogenic potential of HNMT in BC cells. (A-B) MDA-MB-231 cells harboring HNMT siRNA (A) or overexpressing HNMT (B) were subjected to cell viability, migratory wound healing, and anchorage-independent growth soft agar assays. Invasion assay in MDA-MB-231 cells overexpressing HNMT (B). The irregular red lines in the migration assay images indicate wound healing areas. Western blot analysis confirmed the expression of the indicated proteins in MDA-MB 231 cells (231-Wt) and stable cells (231-Vc, 231-HN) (B). (C) On the left are bioluminescence images of 231-Wt-Luc, 231-Vc-Luc, and 231-HN-Luc tumor-bearing mice. Lung and tumor tissues from 231-Vc and 231-HN mice were collected after sacrifice for H&E staining to observe and quantify metastatic nodules. The representative H&E staining image on the right includes high-magnification images of 231-Vc and 231-HN tumor tissues highlighted in blue and red boxes, respectively. (D-E) Western blot analysis confirmed the expression of the indicated proteins in MDA-MB 231 cells (231-Wt), stable cells (231-Vc, 231-HN), primary cells derived from xenografted tumors (231-Vc-XE, 231-HN-XE), metastatic lung cancer cells (231-HN-LM) (D), and 231-HN cells harboring HNMT-scramble or HNMT-siRNA (E). (F) 231-HN and 231-Vc cells underwent RNA sequencing analysis (lower panel). We treated these cells with an E2F1-specific inhibitor (HLM006474, 40 μM for 24 h) and performed Western blot analysis to analyze the protein expression of T-AKT, using actin as a loading control (picture below). (G) We treated cells overexpressing HNMT (231-HN) and control cells (231-Vc) with EGF (100 ng/mL) for 24 h, and then performed RNA-Seq analysis. This data was consistent with previously published Genomic comparisons of PEDERSEN gene sets activated by PI3K/Akt or MAPK pathways in HER2+ tumors (NES = 1.24, P value = 0.04). The data are the means ± SEs. Statistical analysis was performed using a two-tailed unpaired Student's t-test. **P < 0.01 and ***P < 0.001. Scale bars = 250 µm (A, B) and 2 mm (C).