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Additional file 1: Figure. S1. Effect of β-escin on HER2 and p95HER2 expression, corresponding to Fig. 3E, F, H and I in the main text. (A, B) Immunofluorescence analysis of HER2 (red) or normal rabbit IgG (as a negative control) with DAPI (nuclei, blue) in BT474 (A) and JIMT-1 cells (B) after treatment with β-escin (20 and 30 μM, respectively) for 24 h. Intensity profiles represent HER2 expression with green signal fluorescence. (C, D) Immunofluorescence analyses of HER2 (C, green) and ICD-HER2 (D, green, 4B5) or normal rabbit IgG (as a negative control) with DAPI (nucleus, blue) in HER2- and p95HER2-overexpressing MDA-MB-231 cells in the presence of β-escin (20 and 30 μM, respectively) for 24 h. Intensity profiles were analyzed with the histogram tool in the Zen blue software and the horizontal line (white dotted line) indicates 100 intensity units (range of y-axis, 0-250 units). Figure. S2. Influence of β-escin on Ki-67 expression, apoptosis and tumor angiogenesis in vivo, corresponding to Fig. 5I-K in the main text. (A) The effect of β-escin on Ki-67 expression. Sections were immunostained to assess Ki-67 (red) or normal rabbit IgG (as a negative control) with DAPI (blue). Original magnification: × 500. (B) β-escin-induced apoptosis was determined by TUNEL assay. The white arrows indicate the TUNEL-positive apoptotic cells. Label solution without terminal transferase enzyme was used as a negative control. Original magnification: × 500. (C) Tumor angiogenesis was evaluated for each group of xenograft tumors. Tissues were immunostained using an endothelial cell marker CD31 (red) or normal rabbit IgG (as a negative control) with DAPI (blue). Original magnification: × 250. Figure. S3. Effect of β-escin on full-length HER2, ICD-HER2, ALDH1, CD44 and vimentin in JIMT-1 xenograft tumors, corresponding to Fig. 6A-E in the main text. (A, B) β-escin administration resulted in a marked downregulation of full-length HER2 and ICD-HER2 in JIMT-1 xenograft tumors. Tumor tissues were immunostained for full-length HER2 (polyclonal antibody 29D8, green), ICD-HER2 (monoclonal antibody 4B5, green) or normal rabbit IgG (as a negative control) with DAPI (blue). High magnification images were taken using confocal microscopy (original magnification: ×500). Quantitative graphs of signal intensities for full-length HER2 (A, ****p

Additional file 2. Experimental Procedure for the Synthesis of NCT-547.

Additional file 3. Supplementary Figures.

Additional file 1. Materials and Methods.

Gynecologic cancers constitute the fourth most common cancer type in women. Treatment outcomes are dictated by a multitude of factors, including stage at diagnosis, tissue type, and overall health of the patient. Current therapeutic options include surgery, radiotherapy, and chemotherapy, although significant unmet medical needs remain in regard to side effects and long-term survival. The efficacy of chemotherapy is influenced by cellular events such as the overexpression of oncogenes and downregulation of tumor suppressors, which together determine apoptotic responses. Phytochemicals are a broad class of natural compounds derived from plants, a number of which exhibit useful bioactive effects toward these pathways. High-throughput screening methods, rational modification, and developments in regulatory policies will accelerate the development of novel therapeutics based on these compounds, which will likely improve overall survival and quality of life for patients.