Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control