Tomatidine

Analytical standards including tomatidine (Sigma-Aldrich, contains dehydrotomatidine as impurity), solanidine (ChemFaces), solasodine (ChemFaces), α-tomatine (Carbosynth USA, contains dehydrotomatine as impurity), α-solanine (ChemFaces), α-chaconine (ChemFaces), and α-solamargine (ChemFaces) were dissolved in methanol at a concentration of 1 mg ml⁻¹.

Tomatidine (Sigma Aldrich) was solubilized in dimethylsulfoxide (DMSO) at a concentration of 4.5 mM and stored at −20°C until use.

Potassium persulfate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox), sodium carbonate, gallic acid, quercetin, amoxicillin, tomatine, tomatidine, and acetone were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Mueller-Hinton agar, DMSO (dimethyl sulfoxide), and all other nonspecified reagents and solvents were purchased from J.T. Baker (Baker-Mallinckrodt, México).

HeLa (ATCC, CCL-2) cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% non-essential amino acids (NEAA) (all from Invitrogen, NY, USA), at 37°C in 5% CO_2.SiHa (ATCC, HTB-35) and Ca Ski (ATCC, CRL-1550) were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transfection experiments were carried out as previously described[23 (link),24 (link)]. For co-transfection experiments, 10 μg of promoter reporter construct (892pCAT6) was co-transfected with 2 μg of either pcDNA4NLSMTGLI1, p4TO6MTGLI2 or pcDNA4/TO/GLI3 expression constructs[25 (link),26 (link)]. β-gal and CAT assays were performed as previously described[27 (link)]. For imunocytochemistryanalysis, cells were cultured in 24 well dishes and GLI1, GLI2 or GLI3 were co-transfected with pEGFP-C1 (Clontech Laboratories, Mountain View, CA, USA) in ratio 9:1 using Lipofectamine (Invitrogene, NY, USA). For functional analysis of SOX18 protein, cells were transfected as previously described[23 (link)].
For modulation of HH signaling activity, cells were treated with 10 μM cyclopamine (Sigma-Aldrich, St.Louis, MO, USA), 10 μM tomatidine (Sigma-Aldrich, St.Louis, MO, USA), 10 μM purmorfamine (Sigma-Aldrich, St.Louis, MO, USA), or 20 μM GANT61 (Selleckchem, Houston, USA) for indicated periods of time.

Being obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan), the human osteosarcoma U2OS (15-yr-old female) cells and HOS (13-yr-old female) cells were supplemented with 10% FBS and 1% penicillin/streptomycin and then cultured in DMEM and Eagle’s MEM, respectively. The cell cultures were maintained at 37 °C in a humidified atmosphere of a 5% CO2 incubator. Tomatidine was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Tomatidine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Standard stock solution was prepared at a concentration of 2 mg/mL in 80% methanol. The standard solution was serially diluted with 80% methanol to obtain calibration standard solutions at concentrations of 6.25, 12.5, 25, 50, 100, and 200 µg/mL. An HPLC apparatus equipped with a PU-2089 pump, UV-2075 spectrophotometer, and CO-2065 column oven (Jasco, Tokyo, Japan) was used. Chromatographic separation was achieved using an XTerra RP 18 column (4.6 × 150 mm, 5 µm; Waters, Milford, MA, USA) with the column oven temperature maintained at 35 °C. The mobile phase consisted of acetonitrile (Solvent A) and 25 mM triethylammonium phosphate in water (pH 3.0; Solvent B). The mobile phase flow rate was 0.8 mL/min with gradient elution. The initial percentage composition of Solvent A was 20%. It was gradually increased to 45% for 12 min, to 55% for 5 min, and to 57% for 3 min, followed by equilibration to the initial composition for 5 min. All solvents for HPLC analysis were of HPLC grade and were purchased from J.T. Baker (Phillipsburg, NJ, USA).