Cell death detection elisaplus assay

1-(3-Chloro-4-((trifluoromethyl)thio)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (FND-4b) was synthesized and characterized as previously described [28 (link)]. Roswell Park Memorial Institute (RPMI) 1640 Medium and Eagle’s Minimum Essential Medium (EMEM) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM) was from Corning (Corning, NY). Human breast cancer stem cell complete growth medium was from Celprogen (Torrance, CA). MEM non-essential amino acid solution (100x), sodium pyruvate solution (100 mM), insulin solution (10 mg/mL), penicillin-streptomycin (100x) (P/S), and fetal bovine serum (FBS) were from Sigma-Aldrich. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) was from Abcam (Cambridge, MA). Antibodies for pAMPKα (Thr172), total AMPKα, phosphorylated acetyl-CoA carboxylase (ACC), total ACC, phosphorylated ribosomal protein S6, total S6, and PARP were from Cell Signaling Technology (CST; Danvers, MA). The cyclin D1 antibody was from Abcam. The beta-actin antibody was from Sigma-Aldrich. The secondary antibodies to rabbit and mouse were from Santa-Cruz. All relevant antibody information is in Table 1. The Sulforhodamine B (SRB) Cytotoxicity Assay was from G-Biosciences (St. Louis, MO). The Cell Death Detection ELISA^PLUS assay was from Sigma-Aldrich.

Twenty-four hours after cell transfection or not, sensitivity to paclitaxel was measured using either 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) cytotoxicity assay after 72 h exposition to increasing concentrations of paclitaxel, or the Cell Death Detection ELISA^PLUS assay, according to the manufacturer’s instructions (Roche, Sigma) after 24 h paclitaxel treatment. The IC₅₀ was determined with Prism4 software (GraphPad software). Each value represents the mean ± s.e.m. of at least four independent experiments.