Figure 1.

Suppressor-tRNA restores E-cadherin expression in AGS cell line. (a) Schematic representation of the plasmids used: pIRES-EGFP2 vector with a MCS distant from CMV promoter to allow for stable tRNA expression (left side); and from a vector with the sequence of both Sup-tRNA^Arg gene and CDH1 mini-gene (right side). (b) The tRNA_UCG^Arg secondary structure indicating the mutation introduced in the anticodon (left side); the mutant tRNA_UCG^Arg (Sup-tRNA^Arg) is able to decode the PTC as arginine originating a full-length protein (right side). (c, d) AGS cells transiently transfected with pIRES-EGFP2-modified vectors: mock-MCS, 1003int, 1003int_sup-tRNA^Arg, Sup-tRNA^Arg, WTint, WTint_sup-tRNA^Arg, 1003_cDNA, 1003_cDNA_sup-tRNA^Arg, WTcDNA, WTcDNA_sup-tRNA^Arg and parental cells, treated and not treated with Lipofectamine 2000. (c) RNA expression was analysed by RT-PCR for CDH1 transcripts, using GFP expression for transfection control, and GAPDH as endogenous RNA control; protein expression was detected by WB using as endogenous control α-tubulin. (d) Immunocytochemistry in cells transfected with 1003int_sup-tRNA^Arg and 1003_cDNA_sup-tRNA^Arg show E-cadherin expression localized at the membrane, whereas control cells 1003int and 1003_cDNA do not have any expression.