Trizol

Mouse monoclonal antibody for α-tubulin, C23, NF-κB p65 and rabbit polyclonal antibody for IgG, IKKα/β, IκBα, NF-κB p50, NF-κB p65 and goat anti-mouse or anti-rabbit secondary antibody conjugated with horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibody for phosphor-IκBα and rabbit monoclonal antibody for phosphor-IKKα/β were from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-5-LOX antibody was from Novus (Littleton, CO, USA). 5-LOX inhibitors, including MK-886, Nordihydroguaiaretic acid (NDGA); leukotriene B4 and leukotriene B4 receptor antagonist LY29311 were from Cayman Chemical Company (Ann Arbor, MI, USA). Collagenase and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TNF-α and enhanced chemiluminescent HRP substrate (ECL) were from Millipore (Bedford, MA, USA). We purchased RPMI-1640 medium, trypsin and anti-rabbit secondary antibody conjugated with Alexa Fluor 488 from Invitrogen (Carlsbad, CA, USA) and fetal bovine serum (FBS) from Biological Industries (Kibbutz Beit Haemek, Israel). Tri-zol was from MDBIO (Taipei, Taiwan). MMLV Reverse Transcriptase kit was from Promega (Madison, WI, USA). Taqman PCR Master Mix and qPCR probes were from Applied Biosystems/Invitrogen (Foster city, CA, USA).

Total RNA was extracted with TRIzol (MDBio, Taiwan). The cDNA was synthesized using M-MLV transcriptase (Promega, MI, USA). The quantitative PCR was performed using KAPA™ PROBE FAST qPCR Kit (KAPABIOSYSTEMS, Boston, Massachusetts, USA) on an Applied Biosystems StepOnePlus™ Real-Time PCR Systems. The CD44 and integrin β3 mRNA expression were analyzed using Maxima SYBR Green qPCR Master Mix kit (Fermentas, Canada). The CD90 primers were 5′-aggacgagggcacctacac-3′ (sense) and 5′-gccctcacacttgaccagtt-3′ (antisense); the CD133 primers were 5′-aaggcatatgaatccaaaattga-3′ (sense) and 5′-ccaccagag gcatcagaataa-3′ (antisense); the CD13 primers were 5′-ca tccatcagagatggcagac-3′ (sense) and 5′-tgctgaagagatcgtt ctgg-3′ (antisense); the CD24 primers were 5′-atgggcagagcaa tggtg-3′ (sense) and 5′-tggaataaatctgcgtgggta-3′ (antisense); the EpCAM primers were 5′-agttggtgcacaaaatactgtcat-3′ (sense) and 5′-ctcccaagttttgagccatt-3′ (antisense); the HPRT primers were 5′-tgatagatccattcctatgactgtaga-3′ (sense) and 5′-caagacattctttccagttaaagttg-3′ (antisense); the CD44 primers were 5′-tttgcattgcagtcaacagtc-3′ (sense) and 5′-gttacaccccaatcttcatgtccac-3′ (antisense) and the β3 integrin primers were 5′-ccgtgacgagattgagtca-3′ (sense) and 5′-aggatggactttccactagaa-3′ (antisense).

RNA was extracted from tissues using TRIzol (MDBio Inc., Taipei, Taiwan). Synthesis of cDNA was achieved using MMLV RTase (Promega, Madison, Wisconsin, USA). Synthesized cDNA was used as the template for semi-quantitative RT-PCR and real-time quantitative PCR. The primer sequences were as follows: mouse growth hormone (GH): forward, 5′-CAGCCTGATGTTCGGCACCTCGGA-3′ and reverse, 5′-GCGGCGACACTTCATGACCCGCA-3′; mouse IGF-1: forward, 5′-CTGGACCAGAGACCCTTTGC-3′ and reverse, 5′-AGAGCGGGCTGCTTTTGTAG-3′; mouse GAPDH: forward, 5′-GCCATCAACGCCCCTTCATT-3′ and reverse, 5′-ACGGAAGGCCATGCCAGTGAGCTT-3′. Mouse GH (Mm00433590_g1); IGF-1 (Mm00439560_m1); and GAPDH (Mm99999915_g1) TaqMan probes were purchased from Applied Biosystems (USA). The data were analyzed by the StepOne Real-Time PCR system (ABI, USA). The mRNA levels of GH and IGF-1 were normalized to that of GAPDH and expressed relative to the control using the formula 2^-ΔΔCT.