<p>(A) Readthrough efficiencies for APC L360X nonsense mutations were determined in DLD-1 cells in the presence of G418 (10, 25, 50, 100 and 200 µg/ml), negamycin (1 mg/ml), amikacin (2 mg/ml) or gentamicin (800 µg/ml). (B) Readthrough efficiencies for APC R1114X nonsense mutations were determined in NIH3T3 cells in the presence of G418 (50, 100 and 200 µg/ml) or negamycin (1 mg/ml). The readthrough efficiencies in DLD-1 cells were consistent with those in NIH3T3 cells (data not shown). (C) Aminoglycoside treatment restored APC activity. APC binding to beta-catenin (reppression of the pTOPGlow reporter) is restored by the treatment of DLD-1 cells transiently transfected with cDNA APC L360X with G418 (10, 25, 50, 100 and 200 µg/ml), negamycin (1 mg/ml), amikacin (2 mg/ml) or gentamicin (800 µg/ml). (D) APC binding to beta-catenin (repression of pTOPGlow reporter) is restored by the treatment of LoVo cells carrying APC R1114X with G418 (50, 100 and 200 µg/ml) or negamycin (1 mg/ml). As a control, LoVo cells were cotransfected with the pTOPGlow reporter plasmid and either the APC-targeting siRNA (siRNA APC) or a non-targeting siRNA (siRNA NT) and treated with G418 (200 µg/ml). (E) Effect of the siRNA targeting APC mRNA. We transiently transfected LoVo cells with an siRNA targeting (siRNA APC) or not targeting (siRNA NT) APC mRNA. Quantitative PCR was used to determine mRNA levels. Results are expressed relative to the amount of mRNA in the presence of siRNA NT. Mean values are presented, together with the SEM (n = 3).</p
