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. 2013 Aug 16;7(6):1043–1055. doi: 10.1016/j.molonc.2013.08.003

Figure 2.

Figure 2

MREs‐regulated and adenovirus‐mediated TRAIL expression exhibited uveal melanoma specificity. (A) 2 copies of miR‐34a, miR‐137 and miR‐182 MREs were inserted immediately after the stop codon of a TRAIL‐coding open reading frame on a replication‐defect adenoviral vector Ad‐TRAIL to generate Ad‐TRAIL‐3MREs. Ad‐EGFP was used as a control. (B) Normal cells (ARPE‐19 and L‐02) and uveal melanoma cells (SP6.5 and OM431) were treated with Ad‐EGFP, Ad‐TRAIL and Ad‐TRAIL‐3MREs of 10 MOI. Proteins were extracted 48 h after adenovirus infection and detected for TRAIL expression with GAPDH as loading control. (C) Total RNA was also extracted from the same cells. TRAIL mRNA was subsequently quantified by qPCR with GAPDH as endogenous reference. (D) The production of excreted TRAIL protein was also evaluated in the same cells by ELISA assay. qPCR and ELISA assays were both repeated in triplicate and means ± SD were shown. (E) L‐02 and SP6.5 cells were transfected with mixed miRNA mimics or control (30 nM), followed by indicated adenovirus infection (10 MOI). 48 h later, TRAIL expression was determined with GAPDH as endogenous reference by immunoblotting.