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Silencing of MAGE-C1/CT7 expression in SKO-007 MM cell line transduced with the pRS-shRNA-MAGE-C1/CT7 construct.

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posted on 2011-11-16, 00:43 authored by Fabricio de Carvalho, Erico T. Costa, Anamaria A. Camargo, Juliana C. Gregorio, Cibele Masotti, Valeria C.C. Andrade, Bryan E. Strauss, Otavia L. Caballero, Djordje Atanackovic, Gisele W.B. Colleoni

Short hairpin RNA (shRNA) constructs against MAGE-C1/CT7 was stably transduced in MM cell line SKO-007 and effective knockdown was confirmed by qPCR and western blot. A) Schematic representation of the pRETRO-SUPER (pRS) retroviral vector used for silencing of shRNA-MSAGE-C1/CT7 transcribed from the H1-RNA promoter. B) MAGE-C1/CT7 expression was 4-5-fold lower in inhibited cells when compared with wild type, empty vector and ineffective shRNA control cells. Du145 prostate cell line was used as negative control of MAGE-C1/CT7. C) Western blot using anti-MAGE-C1/CT7 monoclonal antibody (clone CT7.33). α/β-Tubulin protein was used as an internal control. Notably, wild type, empty vector and ineffective shRNA control cells showed constitutive expression of MAGE-C1/CT7 protein. MAGE-C1/CT7 protein expression was approximately four times lower in inhibited cells when compared with wild type, empty vector and ineffective shRNA control cells [124 kDa - MAGE-C1/CT7 protein; 55 kDa - α/β-Tubulin protein]. D) Inhibited cells transduced with pRS-shRNA-MAGE-C1/CT7 construct had an approximately 4-fold (70–80%) decrease in MAGE-C1/CT7 protein expression when compared with control cells. Bars represent the densitometric analysis of protein bands normalized to α/β-Tubulin bands presents in Fig. 2C.

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