Gant61

Normal human smooth muscle (HA-VSMC), RMS (RH30, aRMS), (RD, eRMS) and rhabdoid cell line (A204) cells were procured from American Type Culture Collection (ATCC). While (CW9019, aRMS) and (SMS-CTR, eRMS) cells were gifted to JGP by Dr. Frederic G. Barr, Deputy Branch Chief and Senior Investigator Laboratory of Pathology, National Cancer Institute, USA. RMS cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin at 37°C in a humidified atmosphere of 5% CO₂. HA-VSMC were grown in F-12K medium supplemented with 0.05 mg/ml ascorbic acid, 0.01 mg/ml insulin, 0.01 mg/ml transferrin, 10 ng/ml sodium selenite, 0.03 mg/ml endothelial cell growth supplement (ECGS); fetal bovine serum to a final concentration of 10%, HEPES to a final concentration of 10 mM, TES to a final concentration of 10 mM. GANT-61 was purchased from Cayman (MI, USA). Rapamycin was purchased from Fujian Kerui Pharmaceutical Co., Ltd (Fuzhou, Fujian, China). Temsirolimus and vincristine were obtained from Pfizer (NY, USA) and Hospira (IL, USA) respectively. Primers used in this study were obtained from Invitrogen (Supplementary Table S1-I and II). Primary antibodies were purchased as described in supplementary Table S2. Various treatments of these cells were performed when their confluence reached to about 70-80%.

Human colorectal tumor tissue-derived ALI organoids were cultured as described previously [10 (link)]. The medium components were as follows: Advanced Dulbecco’s Modified Eagle’s Medium (DMEM) with 50% Wnt, Noggin and R-Spondin conditioned medium; GlutaMax; B-27 supplement; 100 μg/mL Primocin (Invitrogen, Carlsbad, CA, USA); 1 mM N-Acetyl-l-cysteine; 10 mM Nicotinamide (Sigma-Aldrich, St. Louis, MO, USA); 50 ng/mL mouse epidermal growth factor (EGF) (PeproTech, Inc., Rocky Hill, NJ, USA); 500 nM A83-01 (Adooq Bioscience, Irvine, CA, USA); and 3 μM SB202190; 10 μM Y-27632 (Cayman, Ann Arbor, MI, USA). Stem cell-related signal inhibitors were as follows: YO-01027 (Toronto Research Chemicals, Toronto, Canada); DAPT (Adooq Bioscience); WAV939; Wnt-C59; AY9944; and GANT61 (Cayman). Anti-cancer drugs were as follows: 5-FU (WAKO, Tokyo, Japan); Irinotecan (LC Laboratories, Woburn, MA, USA); and Oxaliplatin (Adooq Bioscience). Antibody sources were as follows: GLI-1 (Gene Tex, Irvine, CA, USA); CD44 (Bethyl Laboratories, Montgomery, TX, USA); c-Myc; and Nanog (Cell Signaling, Beverly, MA, USA). Secondary antibodies were as follows: Horseradish peroxidase (HRP) conjugated anti-rabbit IgG; HRP conjugated anti-goat IgG (Cayman); and HRP conjugated anti-mouse IgG (Millipore, Temecula, CA, USA).

NIH3T3-E1 fibroblasts were obtained from ATCC (Manassas, VA) and cultured, as previously described [35 (link),36 (link)]. CAPAN-1 and PANC-1 human pancreatic cancer cells were obtained from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT, USA) and antibiotics, as previously described [37 (link)]. The human lung cancer cell lines A549 and H2030 were obtained from ATCC and cultured in RPMI-1640 containing 10% FBS. The human hepatoma cell line HepG2 was obtained from ATCC and cultured in DMEM containing 10% FBS. Mouse embryonic fibroblasts (MEFs) from suppressor of fused (Sufu) null mice (Sufu^−/−) were provided by Dr. Philip Beachy of Stanford University and cultured in DMEM containing 10% FBS. Gant61, SB431542 and vismodegib (GDC0449) were obtained from Cayman Chemical, HPI-1 was obtained from Abcam. For experiments, cells were treated in a medium containing 5% FBS.