Z vad fmk

AF, N-acetyl-L-cysteine (NAC), Tertiary butylhydroquinone (Tbhq), glutathione ethylene ester (GEE), ascorbic acid (Vitamin C), Annexin V, propidium iodide (PI) and rhodamine-123 were obtained from Sigma-Aldrich (St. Louis, MO). DCF-DA and z-VAD-fmk were from BD Biosciences (San Jose, CA). Antibodies (Abs) against c-Abl (C-19), ubiquitin (P4D1), Mcl-1 (S-19), caspase-3, -8, -9, apoptosis-inducing factor (AIF), Bcl-2, Bax, GAPDH (FL-335) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against poly (ADP)-ribose polymerase (PARP, clone 4C10-5) was from BD Biosciences. Antibodies against phospho-c-Abl at Y245, phospho-Erk1/2 (T202/Y204), Erk1/2, phospho-Akt, Akt, IκB-α, Bcl-xL, survivin, cleaved caspase-3, -9, cytochrome C, and XIAP were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against phospho-STAT5A/B (Y694/Y699, clone 8-5-2), STAT5, and Ki67 were from Upstate Technology. Enhanced chemiluminescence (ECL) reagents were purchased from Amersham Biosciences (Piscataway, NJ, USA).

PtPT was synthesized in our laboratory and stored as a 20 mM stock solution in dimethyl sulfoxide (DMSO) at −20 °C. Annexin V, propidium iodide (PI) and rhodamine-123 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The proteasome inhibitor, bortezomib (PS341), was purchased from BD Biosciences (San Jose, CA, USA). b-AP15, Suc-LLVY-AMC, Z-LLE-AMC, Boc-LRR-AMC were provided from Boston Biochem (Cambridge, MA, USA). The pan-caspase inhibitor, z-VAD-fmk, was purchased from BD Biosciences. These reagents were dissolved in DMSO as a stock solution, and stored at −20 °C. In all experiments, the final concentration of DMSO did not exceed 0.1%. The metal chelator, ethylenediamine tetraaceticacid (EDTA), was obtained from Sangon Biotech (Shanghai, China). A stored solution (100 mM) was prepared by dissolving EDTA in H₂O and kept at −20 °C.

Stable transfected human T-cell leukemia Jurkat cell lines Jneo and JBcl2 were kindly provided by Dr. Chris Bleackley (University of Alberta), JR cells were kindly provided by Dr. Hannah Rabinowich (University of Pittsburgh). The human normal breast epithelial cell lines MCF-10A was obtained from ATCC and maintained as previously described [42 (link)]. B16-F0 (ATCC, CRL-6322), B16-F10 (ATCC, CRL-6475), Hs578BST (ATCC, HTB-125) and Hs578T (ATCC, HTB-126) cells were obtained from ATCC (Manassas, VA, USA). B16-F0, B16-F10 and Hs578T were maintained in Dulbecco's Modified Eagle's Medium, high glucose, with 10% FCS; Hs578BST was maintained in Dulbecco's Modified Eagle's Medium, high glucose, with 10% FCS and 30ng/ml EGF. Z-VAD-fmk and z-DEVD-fmk were purchased from BD Pharmingen (Mississauga, ON, Canada), SYTOX Green death stain and Alexa Fluor 647 Annexin V conjugate were all obtained from Invitrogen (Carlsbad, CA, USA). All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless indicated otherwise.

TRAIL was commercially supplied (SuperKiller, Enzo Life Sciences, Farmingdale, NY, USA) or produced from 293T cells as described^{30 (link)}. Caspase inhibitors (BD Pharmingen, San Diego, CA, USA) were: Z-VAD-FMK (general caspase inhibitor), Z-FA-FMK (negative control). Bcl-2, Bcl-xL inhibitors ABT-263 and WEHI-539 were purchased from Cayman Chemicals (Ann Arbor, MI, USA). FasL and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (MO, USA). NUTLIN-3a was purchased from MedChemExpress (NJ, USA). Doxycycline was purchased from Sigma-Aldrich (Cat. No: D9891). D-luciferin was purchased from Biotium (CA, USA). Chaetocin was purchased from two sources (C9492–1mg, Sigma-Aldrich, MO, USA) and (S8068, Selleckchem, Houston, TX, USA). The epigenetic tool library was constructed as described^{31 (link)}.

For analysis of apoptosis, the caspase-glo 3/7 assay (Promega) was used according to manufacturer’s instructions. Cells were seeded in white walled 96-wells plates (Corning BV Life Sciences) in densities which resulted in 70% confluence after 24 h as described previously.^{39 (link)} HT1080, JJ012, SW1353, NDCS1 and CH2879 cells were treated with their IC₇₅ of metformin, phenformin, chloroquine and CB-839 (based on 72 h dose response curves). The concentration of compounds used was 10 mM metformin, 100 μM phenformin, 50 μM chloroquine and 6 μM CB-839 if IC₇₅, were above these concentrations. CH2879 cells treated with 1 μM doxorubicin (obtained from the in-house hospital pharmacy) and 50 μM ABT-737 (Catalog No. S1002, Selleckchem) were used as positive control. For the negative control doxorubicin and ABT-737 were combined with Z-vad-FMK (550377, BD Biosciences). After 24 h the caspase-glo substrate was added 1:1 followed by incubation of 60 min at room temperature. Wells were analysed using Wallac 1420 VICTOR2. The experiment was performed two times in duplicate. Data was corrected for plane RPMI control and normalised to untreated control for each cell line. Viability was measured on a simultaneously treated plate after 24 h.

Timed-pregnant female mice were anesthetized with avertin (1.25% v/v, Sigma), and uterine horns were exposed. The bicistronic construct harboring the gene-of-interest and an internal ribosome entry site (IRES)-GFP cassette in the pCAGIG expression vector (1–2 μg μl⁻¹ at 1 μl volume) in endotoxin-free water containing Fast Green (1:10,000, Roth) was electroporated into the lateral ventricle of the embryo via a glass capillary at embryonic day 14.5. Electroporation was performed with tweezer electrodes (5 pulses of 40 V for 50 ms at 950 ms intervals) using an SP-3c electroporator (Supertech). After electroporation, the uterine horns were returned into the abdominal cavity, the wall and skin were sutured and embryos were allowed to continue their normal development. To inhibit caspase activity, the pan-caspase inhibitor Z-VAD-FMK (5 μM, BD Biosciences) was injected in a similar manner as above. To identify proliferating cells, BrdU in 0.9% saline (200 mg kg⁻¹, Sigma) was intraperitoneally injected into pregnant dams at E14.5. The embryos were collected 2 h later and their brains were fixed with 4% paraformaldehyde (PFA).

2 × 10⁶ MCF7 cells were seeded in 25 cm² flasks. After 24 h, cells were pre-treated with 50 μM of the pan-caspase inhibitor Z-VAD-FMK (BD Pharmingen) or 100 nM wortmannin (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h before co-incubating them with 25 μM of compound 5, 30 μM of compound 15 or vehicle (maximum amount of DMSO used in the treatments) for 48 h. Cells were collected and processed with the Apo-Direct kit as described previously.