Azd0530

All animal work adhered to the Agency for Science, Technology and Research (A*STAR; Singapore), Institutional Animal Care and Use Committee (IACUC) guidelines on the use and handling of animals. SKOV3-Luc-D3 cells (Xenogen Co., Alameda, CA, USA) at a density of 3.5×10⁶ in 100 μl of PBS were injected into the intraperitoneal cavity of 4-week-old female BALB/c nude mice. At 6 weeks post-implantation, the mice were randomly divided into control and treatment groups (n = 5 animals per group). For the treatment group, mice were administered via oral gavage with 50 mg/kg AZD0530, 50 mg/kg BIBF1120, or 25 mg/kg AZD0530 plus 25 mg/kg BIBF1120 (Selleck Chemicals, Houston, TX) for 5 days a week for 2 weeks. The drug was re-suspended in 0.5% hydroxypropyl methycellulose (Sigma-Aldrich) and 0.1% polysorbate buffer (Sigma-Aldrich). The control group received the vehicle buffer alone. The growth of tumor xenografts was monitored by bioluminescence using the IVIS system 2000 series (Xenogen Co.). The xenografts were harvested at 8 weeks post-implantation for gene expression microarray analysis (Affymetrix GeneChip Human Gene 1.0ST Array, Santa Clara, CA, USA) to ascertain the EMT scores, and then subjected to paraffin embedding followed by immunohistochemical staining for E-cadherin (#3195S, Cell Signaling Technology, Beverly, MA).

MCF-10A-ER-Src cells (Iliopoulos et al., 2009 (link); Iliopoulos et al., 2010 (link); Ji et al., 2018 (link); Ji et al., 2019 (link)) were grown in DMEM/F12 without phenol red (Thermo Fisher Scientific, 11039–047) + 5% charcoal stripped FBS (Sigma, F6765) + 1% pen/strep (Thermo Fisher Scientific, 15140122) + 20 ng/ml EGF (Peprotech, AF-100–15) + 0.5 μg/ml Hydrocortisone (Sigma, H-0888) + 0.1 μg/ml cholera toxin (Sigma, C-8052) + 10 μg/ml insulin (Sigma, 10516). 1–0.4 μM Tamoxifen (Sigma, H7904) + 2–4 μM AZD0530 (Selleck Chemicals, S1006) were used to induce the transformation. MDA-MB-231 cells were grown in DMEM (Thermo Fisher Scientific, 11995–073) + 10% FBS (Sigma, TMS-013-B) + 1% pen/strep.

Cells were seeded for 24 h and then transfected with siRNAs, 50 nM (Dharmacon) and Lipofectamine RNAiMax (Life Technologies). siRNA sequences were in Supplementary Data 3. After 24 h, cells were split and treated with either ethanol or Tamoxifen (0.4 µM, Sigma-Aldrich, H7904) plus AZD0530 (0.4 nM, Selleck Chemicals, S1006) for 24 h. Total RNA was isolated using mRNeasy Mini Kit (Qiagen, No. 217004). Two microgram RNA was used for SYBR Green based. Primers were listed in Supplementary Data 4.