MV4-11, MOLM-13, and MOLM-14 are 3 AML cell lines. MV4-11 has a 48, XY, t(4;11)(q21;q23), þ8, þ19 karyotype with a 30-bp FLT3-ITD mutation. MOLM-13 has the 51(48-52)<2n>XY, þ8, þ8, þ8, þ13, del(8)(p1?p2?), ins(11;9)(q23;p22p23) karyotype, carrying KMT2A-MLLT3 mutations and a 21-bp FLT3-ITD mutation. Finally, MOLM-14 has the -49(46-50)<2n>XY, þ6, þ8, þ13, der(2)t(1;2) (q31;q35), ins(11;9)(q23;p22p23), del(14(q23q32.3), del(16) (q11.2q13.1) karotype, including also KMT2A rearrangement and a 21-bp FLT3-ITD mutation. All AML and BMSC lines (MV4-11, MOLM-13, MOLM-14, MS5) were cultured in an a-minimum essential medium (aMEM), supplemented with 10% FCS, 2 mmol/L L-glutamine, 50 U/mL penicillin, and 50 mg/mL streptomycin. Hypoxia at 1% was induced by incubating cells in a specific oxygen (O 2 ) chamber (BioSpherix). Stromal cells were implanted for 3 days to reach 80% confluence, then culture medium was removed, and leukemic cells were added to the flask. Coculture cells were incubated either in a specific O 2 chamber for 1% O 2 hypoxic culture or in an incubator at 5% CO 2 for normoxic culture. Cells were cultured with vehicle or gilteritinib (ASP2215; kindly provided by Astellas Pharma) or quizartinib (AC220; LC Laboratories), both dissolved in DMSO.
Ac220
Manufactured by LC Laboratories
The AC220 is a power supply designed to provide a stable and reliable source of alternating current (AC) at 220 volts. It is a versatile piece of equipment that can be used in a variety of laboratory and industrial applications that require a consistent and regulated AC power supply.
Automatically generated - may contain errors
Lab products found in correlation
Sorafenib, by LC Laboratories (1 mentions)
Cep 701, by LC Laboratories (1 mentions)
Pkc412, by LC Laboratories (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Lamtor2, by Cell Signaling Technology (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Okadaic acid, by Cayman Chemical (1 mentions)
Sorafenib, by Selleck Chemicals (1 mentions)
Sunitinib, by Selleck Chemicals (1 mentions)
Ruxolitinib, by Selleck Chemicals (1 mentions)
Fty720, by Cayman Chemical (1 mentions)
4 protocols using ac220
1
Comparative Analysis of AML Cell Line Responses to FLT3 Inhibitors
2
Compound Preparation for Cancer Research
3
Phosphorylation Analysis of Key Signaling Proteins
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For phosphorylation analysis of AKT, mTOR, p70 S6 Kinase 1, S6 ribosomal protein and ERK1/2, cells were stimulated for 20 min with Flt3 ligand. In addition, where stated, cells were treated with Rapamycin (50 ng ml−1, LC Laboratories) or AC220 (100 ng ml−1, LC Laboratories) for 1 h, before stimulation. Cell lysates of BMDCs and splenic DCs were prepared2 (link) and separated on a SDS–polyacrylamide gel electrophoresis. Membranes were probed after blotting and blocking against primary antibodies (LAMTOR2, LAMTOR3 (ref. 10 (link)), pAKT (1:500, clone D9E), AKT (1:500–1,000), pp70S6K1 (1:1,000, clone 1A5), pERK1/2 (1:500, clone E10), ERK1/2 (1:1,000, clone 137F5), pS6 (1:1,000, clone D57.2.2E) from Cell Signaling; Actin (1:5,000, clone C4) from Millipore; LAMTOR1 (1:1,000), LAMTOR4 (1:1,000) from Atlas; LAMTOR5 (1:500, clone C16) from Santa Cruz) overnight at 4 °C in blocking buffer (3% BSA, 1 mM EDTA, pH8, 0.05% Tween 20, 6 mM Sodium Azide, pH 5.2). Membranes were washed with Tris -buffered saline (50mM Tris-HCl, pH 7.6; 150mM NaCl) with 0.05% Tween 20 and incubated with the secondary antibodies (anti-mouse-/anti-rabbit IgG peroxidase antibody, 1:5,000 (Sigma)). Detection was performed by chemiluminescence. Uncropped western blots are provided in Supplementary Figs 7 and 8 .
4
Comparative Analysis of Kinase Inhibitors
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FTY720, CEP701, PKC412 and okadaic acid (OA) were from Cayman Chemicals; sorafenib, sunitinib and ruxolitinib from Selleckchem; AC220 from LC Laboratories; and AAL(S) was synthesized as previously described [58 ]. The chemical structures of all drugs are shown in Supplementary Figure S8 .
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