A P-value below 0.01 was considered significant. Acknowledgments This work was partially funded by European Regional Development Fund, European Territorial Cooperation 2007-2013, CCI 2007 CB 163 PO 037, OP Italia-Malta 2007-2013. Irvine, CA, USA). Then, after removal of residual genomic DNA with DNase I (Zymo Study), oligo (dT)-primed reverse transcription was performed on 1?g of total cellular RNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories Srl, Milan, Italy), following a manufacturer’s instructions. For real-time PCR analyses, each cDNA sample was amplified by IQ SYBR Green Supermix (Bio-Rad Laboratories), as reported previously,57 using the QuantiTect primers Oct3/4, Sox2, Nanog58 (Qiagen, Milan, Italy). All PCR reactions were performed in triplicate in 96-well plates; each reaction mixture contained 2?l of template cDNA, 10?l of SYBR Green PCR Expert Blend 2X (Bio-Rad Laboratories), ahead and reverse primers in the concentration of 300?nM and RNase-free dH2O to a final volume of 20?l. Reactions were performed in iQ5 Thermal Cycler Instrument (Bio-Rad Laboratories), as reported previously.58 The relative quantities of analyzed genes were determined using the 2CCt method and the data were normalized with the endogenous control, GAPDH (Qiagen). Western blotting analysis Cell lysates and protein samples were prepared as reported previously.57 Equal amounts of protein samples Apoptosis Inhibitor (M50054) (50?g per lane) were run inside a SDS-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane. All analyses were performed using specific primary antibodies, which were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Then, the detection was developed by using a secondary antibody conjugated with alkaline phosphatase. Protein bands were visualized using nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl-phosphate NUDT15 (Promega, Milan, Italy) and their intensity was quantified by densitometric analysis using the SMX Image software (Bio-Rad Laboratories). The correct protein loading was ascertained by reddish Ponceau staining and immunoblotting for -actin. All the blots demonstrated are representative of at least three different experiments. Statistical analysis Results are offered as meanS.D. of data from at least three self-employed experiments. Data were analyzed using Student’s t-test. A P-value below 0.01 was considered significant. Acknowledgments This work was partially funded by Western Regional Development Account, Western Territorial Assistance 2007-2013, CCI 2007 CB 163 PO 037, OP Italia-Malta 2007-2013. Drs. G Apoptosis Inhibitor (M50054) Buttitta, R Di Fiore and R Drago-Ferrante benefit by contract grants supported from the same above-mentioned Western Account Italia-Malta 2007-2013. Dr. D Carlisi is definitely a recipient of a give by Italian Ministry of Education, University or college and Study’ (MIUR). Apoptosis Inhibitor (M50054) Glossary BAPTA-AM1,2-bis-(o-aminophenoxy)-ethane-N,N,N‘,N-tetraacetic acid, tetraacetoxymethyl esterBCIP5-bromo-4-chloro-3-indoyl-phosphateCSCcancer stem cellDETCdiethyldithiocarbamateDHEdihydroethidiumDMAPTdimethylaminoparthenolideDPIdiphenylene iodiniummmitochondrial membrane potentialFITCfluorescein isothiocyanateH2-DCFDA5-(and-6)-carboxy-2,7-dichlorodihydrofluorescein diacetateHPFhydroxyphenyl fluoresceinJC-15,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodideMnSODmanganese-dependent superoxide dismutaseNACN-acetylcysteineNOXNADPH oxidaseNrf2nuclear element erythroid 2-related element 2PDTCpyrrolidine dithiocarbamatePIpropidium iodidePNparthenolideROSreactive oxygen speciesSAHAsuberoylanilide hydroxamic acidSFsulforaphanetBHQtert-butylhydroquinoneTNBCtriple-negative breast cancer Notes The authors declare no discord of interest. Footnotes Edited by G Melino.