Q vd oph

MCF-7 and MDA-MB231 cell lines were from American Type Culture Collection (ATCC, Rockville, USA), and Cal51 from DSMZ (Braunschweig, Germany). The ER-Src MCF-10A cell line was a generous gift of Dr Kevin Struhl (Harvard Medical School, Boston, USA). All cell lines were cultured following supplier's recommendations. The ER-Src MCF-10A cells contain an integrated fusion of the v-Src oncoprotein and the ligand-binding domain of estrogen receptor and are induced to rapidly transform when grown with 1 μM 4OH-TAM (Sigma-Aldrich, Saint-Quentin Yvelines, France), as previously described [13 (link)].
YM155, Necrostatin-1 and BAY11-7085 were purchased from Selleck Chemicals (Houston, USA) and the pan-caspase inhibitor Q-VD-OPh from R&DSystems (Abingdon, UK). Chloroquine, 3-Methyl-Adenin (3-MA), Bafilomycin A1 were purchased from Sigma-Aldrich (Saint-Quentin Yvelines, France). AS602868 was a generous gift of Merck-Serono International SA [14 (link)]. Antibodies against Survivin was purchased from R&DSystems (Lille, France), antibodies against LC3, p21, pS15-p53, pS317-Chk1, pT68-Chk2, IKK2, Actin, γH2AX from Merck Millipore (Saint-Quentin en Yvelines, France), antibodies against HSP90, p53 and cleaved (i.e. activated) caspase3 from Becton Dickinson (Pont de Claix, France), antibody against BAX from Dako (Courtaboeuf, France), and antibody against p62 from Santa Cruz (Heidelberg, Germany).

N-(p-amylcinnamoyl)anthranilic acid (ACA), maintained as a 50 mM stock solution in dimethylsulfoxide (DMSO), 2-aminoethoxydiphenyl borate (2-APB; 75 mM stock solution in DMSO), aristolochic acid (75 mM stock solution in DMSO) and 3-methyladenine (3-MA; 50 mM stock solution in DMSO) were purchased from Sigma (St. Louis, MO, USA). N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG; 0.5 M stock solution in DMSO) was purchased from AccuStandard (New Haven, CT, USA). Doxorubicin hydrochloride and tamoxifen citrate were purchased from Thermo Scientific (Waltham, MA, USA). Q-VD-OPh was purchased from R&D Systems (Minneapolis, MN, USA) and maintained as a 50 mM stock solution in DMSO. Propidium iodide (PI) (10 mg/ml solution) was purchased from Thermo Scientific. ApoScreen Annexin V-fluorescein isothiocyanate (FITC) was purchased from Southern Biotech (Birmingham, AL, USA).

The synthesis of racemic fluorizoline was performed as previously described [2 ]. Actinomycin D was purchased from Enzo Life Sciences (Farmingdale, New York, USA). Q-VD-OPh and Z-IETD-FMK were from R&D systems (Minneapolis, Minnesota, USA). Recombinant mouse TNFα was from PeproTech (Rocky Hill, New Jersey, USA). MnTBAP, etoposide and cycloheximide were from Sigma-Aldrich (Saint Louis, Missouri, USA).

Hydralazine hydrochloride, procainamide, dihydroethidium (DHE) and mouse anti-β-actin monoclonal antibody were from Sigma-Aldrich (ST. Louis, MO). The fluorescent cationic lipophilic dye 3,3′-dihexyloxacarbocyanine iodide (DiOC₆ (3)) and Alexa Fluor 488-labeled goat anti-mouse were obtained from Molecular Probes (Carlsbad, CA). Manganese-porphyrin Mn(III)TMPyP was from Cayman Chemical (Ann Arbor, MI). Q-VD-OPh, a wide-spectrum caspase inhibitor, and anti-human caspase-9 monoclonal antibody were from R&D Systems (Minneapolis, MN). Cytofix/cytoperm was from BD Biosciences (San José, CA). Anti-Bak (Ab-1) monoclonal antibody and anti-Chk1 (pSer317) rabbit polyclonal antibody were obtained from Calbiochem (Darmstadt, Gemany). Anti-phospho-histone H2AX (Ser139) monoclonal antibody was from Upstate/Millipore (Billerica, MA). Anti-human caspase-8 monoclonal antibody was purchased from Cell Diagnostica (Munster, Germany). Anti-human caspase-3 polyclonal antibody was obtained from Stressgen (Ann Arbor, MI). Mouse monoclonal DNMT1 antibody was from Abcam (Cambridge, UK).

Cells at a concentration of 2 × 10⁶ cells/ml per condition were incubated with H₂O₂ (Thermo Fisher Scientific), Q-VD-OPh (R&D Systems) or combinations of these at 37 °C on a shaking heat block for 4 h. Eol-1 cells were pelleted by centrifugation at 3000 × g for 60 s and resuspended with whole-cell lysis buffer. Sample was incubated on ice for 10 min then NP-40 was added, briefly vortexed and centrifuged for 20 min at 3000 × g. Supernatant was removed and the remaining cell pellet was resuspended in sample buffer before boiling at 95 °C for 5 min. Lysate were run on 12% precast gels (Thermo Fisher Scientific, Rockford, IL, USA) and transferred onto PVDF (Immobilon-P, Millipore, Herts, UK). Membranes were blocked for 1 h in 5% (wt/vol) dried milk/TBS/0.1% Tween-20 before probing with antibodies to cleaved caspase-3 diluted 1 : 500 (Cell Signaling Technologies) at 4⁰C overnight or GAPDH diluted 1 : 20 000 (Sigma-Aldrich, St. Louis, MO, USA) 1 h at room temperature. Following 3 × 5 min washes in TBS/0.1%Tween-20, the blots were incubated with HRP-conjugated secondary antibody (Dako, Glostrup, Denmark) diluted 1 : 2500 for 1 h at room temperature before incubation with ECL (GE Healthcare, Bucks, UK) exposure to BioMax MS-1 X-ray-sensitive film, and processing (X-Ograph Imaging Systems, Wilts, UK).