Deferoxamine mesylate dfo

X-11, DODHA (deoxy-dihydroartemisinin), and DOX-11 (10-O-[4-(1-acetyl-5-phenyl-4,5-dihydropyrazol-3-yl) phenyl]-(10S)-deoxy-dihydroartemisinin) were synthesized as described in supp Fig. 3. DHA, acridine orange (AO), ethidium bromide (EB), N-acetylcysteine (NAC) and catalase (CAT), ferrous sulfate (Fe²⁺), ferric ammonium citrate (Fe³⁺), diphenyleneiodonium chloride (DPI) and deferoxamine mesylate (DFO) were purchased from Sigma Chemical Co. (St. Louis, MO). ABT-737 was purchased from Selleckchem (Houston, TX). 5,6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), Rhodamine-123 (Rh123) and MitoSOX™ Red were purchased from Molecular Probes (Eugene, OR). Antibodies to poly-(ADP-ribose)-polymerase (PARP), caspase-3 and caspase-8 were obtained from BD Biosciences (San Diego, CA); to Mcl-1, Bcl-2, Bcl-xL, Bax, and β-actin were from Santa Cruz Biotechology, Inc. (Santa Cruz, CA); to Bim, Bak, CHOP, FOXO3a, Puma, cleaved caspase-9 and -3 were from Cell Signaling Technology, Inc. (Beverly, MA); and to Noxa was from Abcam Inc. (Cambridge, MA).

Using dimethylsulfoxide (DMSO) treated cells as controls, cells were treated with Temsirolimus (TEMS, #50-811-7, Fisher Scientific, Pittsburgh, PA) at a final concentration of 2μM, as described in our prior work (17 (link)). Likewise, as earlier described (24 (link)), 5-Azacytidine (AZA, #S1782, Selleck Chemicals, Houston, TX) was utilized at a final concentration of 1μM while Suberoylanilide hydroxamic acid (SAHA, #S1047, Selleck Chemicals, Houston, TX) was utilized at a final concentration of 50μM. Lysophosphatidic acid (LPA, as a sodium salt in chloroform, #857130C, Avanti Polar Lipids, Alabaster, AL) was utilized at a final dose of 10μM, as previously described (17 (link)). D-erythro-Sphingosine-1-Phosphate (S1P, #860492, Avanti Polar Lipids, Alabaster, AL) was utilized at a final concentration of 250nM. Deferoxamine mesylate (DFO, #D9533, Sigma-Aldrich, St. Louis, MO) was utilized at a final concentration of 10μM, as described in our earlier work (25 (link)).

A mouse breast cancer cell line (4T1) and endothelial cells (SVEC) were grown as monolayer cultures in RPMI-1640 medium supplemented with 1% antibiotic–antimycotic mix and 10% fetal bovine serum (FBS). Cells were infected by retroviruses with reporter gene vectors pCMV-luc2/miR-210 in which luciferase signals could be turned off by the binding of miR-210 to triplicates of the miR-210 binding site at the 3′ end of luc2 mRNA. Stable cell lines were selected by treatment with puromycin (2 g/mL) for 2 weeks. 4T1 cell lines with pCMV-luc2/miR-210 were labeled 4T1/miR210. Hypoxic conditions were induced with deferoxamine mesylate (DFO) (Sigma-Aldrich, St Louis, MO, USA) at 37°C in a 5% CO₂ humidified environment.

B. subtilis colonies were harvested after 24 h and sonicated at 30% amplitude for 30-60 s until homogenous. The cells suspension was then mixed with 2’,7’-dichlorodihydrofluorescin diacetate (DCFH₂-DA; purity ≥97%, Sigma), a fluorogenic dye that is converted to the fluorescent DCF, at a final concentration of 100 µM and incubated in the dark for 1 h. Deferoxamine mesylate (DFO; purity ≥92.5%, Sigma) and N,N,N’,N’-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN; purity ≥97%, EMD Millipore). Cells were pellets by centrifugation at 16,200 × g for 1 min. The supernatant was collected (100 µL) and cells were resuspended in PBS before being were transferred into a 96-well plate (100 µL), and the fluorescent intensity was measured with a TECAN Spark monochromator-based (SparkControl version 3.1) with an excitation wavelength of 485 nm and emission wavelength of 535 nm, bandwidth 20.

The sample size required for animal experiments was based on preliminary data.
For DFO treatment, daily 100mg/kg deferoxamine mesylate (DFO; Sigma) was administered I.P. from day 8 until day 22 post-transplantation of AML blasts, at which time mice were sacrificed and their BM analyzed. Control mice were injected I.P. with 100μl of PBS.
Induction chemotherapy for AML was administered when BM infiltration was over 50% by injecting 100mg/kg cytarabine (Ara-C) I.V. for 5 days and 3mg/kg doxorubicin (Doxo) for 3 days. Ara-C was co-delivered with Doxo on days 1 to 3 and alone on days 4 and 5, mimicking the 7+3 regimen used in AML patients (Wunderlich et al., 2013 (link)). Both drugs were purchased from Sellekchem, MA or obtained from the Imperial College Healthcare NHS Trust.
For EC-specific deletion of Fbxw7, tamoxifen (500 μl/mouse I.P.; Sigma) was given daily to Fbxw7^lox/lox Cdh5(PAC)-CreERT2^T/+ (Fbxw7^iΔEC) mice and to control Fbxw7^lox/lox and WT mice. In experiments where relapse and survival were analyzed, tamoxifen was given daily between day 10 and 20 post-transplantation, at which point chemotherapy was initiated. Blinding was done for the tamoxifen studies.