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

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.