Lipid Raft-Mediated Akt Signaling As a Therapeutic Target in Mantle Cell Lymphoma
SUPPLEMENTARY INFORMATION
Lipid raft-mediated Akt signaling as a therapeutic target in mantle cell lymphoma
Mariana Reis-Sobreiro,1 Gaël Roué,2 Alexandra Moros,2 Consuelo Gajate,1 Janis de la Iglesia-Vicente,1 Dolors Colomer,2 and Faustino Mollinedo1,*
1Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain
2 Hematopathology Unit, Department of Pathology, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, E-08036 Barcelona, Spain
Supplementary Materials and methods
Antibodies
Proteins were identified following Western blot analysis using the following antibodies: anti-60 kDa Ser473 p-Akt; anti-60 kDa Thr308 p-Akt; anti-p-Akt substrate motif (RXRXXS/T); anti-54 and 46 kDa p-JNK 2 and 1; anti-289 kDa Ser2448 p-mTOR; anti-mTOR; anti-85 kDa PI3K; anti-68-58 kDa Ser 241 p-PDK1; anti-68-58 kDa PDK1(1:1000 dilution in TBST with 5% BSA); anti-23 kDa Ser136 p-Bad; anti-15-20 kDa p-4E-BP1; anti-4E-BP1(1:3000 dilution in TBST with 5% BSA) rabbit polyclonal antibodies (Cell Signaling); anti-60 kDa Akt1/2/3; anti-48kDa Fas/CD95 (C-20) rabbit polyclonal antibodies (Santa Cruz Biotechnology) (1:1000 and 1:500 dilution, respectively, in TBST with 5% BSA); anti-42 and 44 kDa p-ERK1 and 2 mouse monoclonal antibody; anti-ERK2 mouse monoclonal antibody; anti-JNK1 rabbit polyclonal antibody (Santa Cruz Biotechnology) (1:1000 dilution in TBST with 5% BSA); anti 23kDa-Bad mouse monoclonal antibody (BD Transduction Laboratories) (1:1000 dilution in TBST with 5% BSA); anti-caspase-9 rabbit polyclonal antibody (Calbiochem) (1:1000 dilution in TBST) that recognizes the cleaved active caspase fragments; anti-42 kDa β-actin monoclonal antibody (Sigma) (1:5000 dilution in TBST).
Xenograft mouse model
Animal procedures in this study complied with the Spanish (Real Decreto RD1201/05) and the European Union (European Directive 2010/63/EU) guidelines on animal experimentation for the protection and humane use of laboratory animals, and were conducted at the accredited Animal Experimentation Facility (Servicio de Experimentación Animal) of the University of Salamanca (Register number: PAE/SA/001). Procedures were approved by the Ethics Committee of the University of Salamanca.Female CB17-severe combined immunodeficient (SCID) mice (Charles River Laboratories), kept and handled according to institutional guidelines, complying with Spanish legislation under a 12/12 h light/dark cycle at a temperature of 22ºC, received a standard diet and acidified water ad libitum. CB17-SCID mice were inoculated subcutaneously into their lower dorsum with 107Z-138 cells in 100 μl PBS and 100 μl Matrigel basement membrane matrix (Becton Dickinson). When tumors were palpable, mice were randomly assigned to cohorts of 7 mice each, receiving a daily oral administration, for a 21-day period, of edelfosine (30 mg/kg body weight), perifosine (26.45 mg/kg), or an equal volume of vehicle (water). The shortest and longest diameter of the tumor were measured with calipers at the indicated time intervals, and tumor volume (cm3) was calculated using the following standard formula: (the shortest diameter)2 x (the longest diameter) x 0.5. Animals were killed, according to institutional guidelines, when the diameter of their tumors reached 3-4 cm or when significant toxicity was observed. Animal body weight and any sign of morbidity were monitored. Mice were killed 24 h after the last drug administration, and then tumors were extirpated, measured and weighed.
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