Gant58

The immortalized human leiomyoma cell line (HuLM) and immortalized human uterine smooth muscle (UTSM) cells were a generous gift from Professor Darlene Dixon. The cells were cultured and maintained in phenol red-free, 10% fetal bovine serum Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12. The leiomyosarcoma (LMS) cell line (SK-UT1, ATCC^® HTB-114^TM) (ATCC, Manassas, VA, USA) was cultured and maintained in ATCC-formulated Eagle’s Minimum Essential Medium with 10% of fetal bovine serum. We used these three cell lines covering the spectrum from a normal cell line (UTSM), benign uterine tumor cell line (HuLM), and uterine malignant cell line (LMS) to better understand the tumor progression linking to the HH pathway.
SMO inhibitors LDE225 and GDC0449 were purchased from Selleck Chemical (Houston TX, USA), GLI inhibitors Gant58 and Gant61 from Sigma Aldrich (St. Louis, MO, USA) and DNA methylation inhibitor 5′ Aza- 2′-deoxycytidine from Biosynth & Carbosynth (Staad, St. Gallen, Switzerland). The range of doses tested was 0.1–60 µM.

All drug treatments were carried out in serum free DMEM/F12 for 24 hours. TGFβ and Sclerostin (R&D Systems) were used at 10ng/ml and 1-2ug/ml respectively. TGFβ buffer (5%BSA in 4mmol HCl) was used as a control. GANT58 and Lithium Chloride (Sigma-Aldrich) were used at 10μM and 20mM respectively. Purmorphamine and SIS3 (EMD Millipore) were used at 10μM. Cyclopamine (LC Labs) was used at 12nM. SB202190 (Tocris) was used at 10μM. VU-WS113, a less cytotoxic derivative of Pyrvinium, was a gift from Dr. Ethan Lee at Vanderbilt University.

SAG, cytochalasin D, 5Z-7-Oxoeaneol, GANT58, EGCG and MG132 were purchased from Sigma, vismodegib from Selleck Chemicals, 20(S)-hydroxycholesterol (20(S)-OHC) from Cayman Chemicals, Bodipy-Cyclopamine from Toronto Research Chemicals, and vinblastine and AZ-TAK1 from Santa Cruz Biotech.