5 fluorouracil

Mice containing the Sin3B-flox (Sin3B^F) allele have been previously described. To generate hematopoietic specific deletion of Sin3B, Sin3B^F/F mice were intercrossed to Vav1-iCre mice, which is active at embryonic day 11.5 (E11.5). Ptprc^a; Pepc^b (CD45.1) congenic mice were purchased from The Jackson Laboratory and bred in-house to use as recipients for competitive transplantation experiments. All mice were kept on an inbred C57BL/6 background. Mice were housed in pathogen-free barrier facilities with a 12-h light/dark cycle and given food and water ad libitum. Mice were administered 5-Fluorouracil (Invivogen) via intraperitoneal injection at 100 mg/kg body weight. For competitive transplantation assays, recipient mice at 8–10 weeks of age were lethally irradiated (total body irradiation) with 12 Gy of γ-irradiation with a MultiRad 350 X-Ray Irradiator (Faxitron^®). Mice were given 2 doses of 6 Gy of irradiation at least 3 h apart. Mice were maintained on sterile, acidified water supplemented with Sulfamethoxazole and Trimethoprim for 2 weeks following irradiation, replenishing the antibiotics after a week. Equal numbers of male and female mice were used in all experiments unless specified otherwise. All animal experiments and protocols were approved by the New York University Grossman School of Medicine Institutional Animal Care and Use Committee.

Unless otherwise stated, all chemicals were purchased from Sigma (St Louis, MO, USA). Stock solutions of amitriptyline (100 mM), bupropion (100 mM), citalopram (10 mM), EGTA (50 mM), fluoxetine (10 mM), and N-acety-l-cysteine (NAC, 300 mM) were prepared in distilled water (DW). 5-fluorouracil (100 μg/mL), doxorubicin (2 mg/mL), paroxetine (20 mM), PD98059 (20 mM), SB203580 (20 mM), SP600125 (20 mM), taxol (1 mg/mL), tianeptine (100 mM), and Z-VAD-FMK (10 mg/mL, InvivoGen, San Diego, CA, USA) were dissolved in dimethyl sulfoxide (DMSO). The solutions were then diluted in culture medium to the working concentration. Where DMSO was used as a solvent, solution containing equivalent concentration of DMSO was used as a control. Final DMSO concentrations for chemicals indicated above were ~0.1% (v/v).
Dulbecco’s modified Eagle’s medium (DMEM/F12), DMEM, fetal bovine serum (FBS), and horse serum were purchased from Gibco-BRL (Gaithersburg, MD, USA). RPMI-1640 was purchased from Lonza (Walkersville, MD, USA). Total glutathione assay kit was purchased from Assay Designs (Ann Arbor, MI, USA). FLICA apoptosis detection kit was purchased from Immunochemistry Technologies (Bloomington, MN, USA). MitoSox was purchased from Invitrogen (Carlsbad, CA, USA). JC-1 mitochondrial membrane potential detection kit was purchased from Biotium Inc (Hayward, CA, USA).

Stocks of retroviruses were generated using pMIGR1 and MIGR1-Bcl-xL retroviral vectors (gift from W. Pear and D. Allman, University of Pennsylvania, Philadelphia, PA; Pear et al., 1998 (link)), as previously described (Schweighoffer et al., 2013 (link)). Syk^fl/+MCM or Syk^fl/−MCM mice were injected intraperitoneally with 100 mg/kg 5-fluorouracil (InvivoGen). 5 d later, BM was harvested, treated with ACK lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, and 100 µM EDTA), then cultured overnight in DMEM-plus (DMEM with 10% FCS [Lonza], 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µM nonessential amino acids, 20 mM Hepes buffer, and 100 µM 2-mercaptoethanol) containing 100 ng/ml rmSCF (PeproTech), 6 ng/ml IL-3 (Sigma-Aldrich), and 10 ng/ml rmIL-6 (PeproTech). 24 h later, cells were moved onto retronectin-coated plates (Clontech T100B; Takara Bio Inc.), and viral supernatant was added and cultured overnight. This infection step was repeated the next day before cells were harvested and injected intravenously (at least 2.5 × 10⁵ cells/recipient) into RAG1-deficient recipient mice that had received 5 Gy irradiation. 6 wk after cell transplantation, the efficiency of infection was assessed by flow cytometry of peripheral blood, and tamoxifen treatment was started as described above.