Identification of APC nonsense mutations responsive to aminoglycoside treatment.

<p>Readthrough efficiencies for 11 nonsense mutations in the APC gene were assessed in NIH3T3 cells with and without gentamicin (800 µg/ml) treatment for 24 h. Two nonsense mutations (L360X and R1114X) displayed levels of gentamicin-induced readthrough of more than 0.5%. Means values are presented, together with the standard error of the mean (SEM) (n = 5).</p

Western blot.

<p>LoVo cells were left untreated or were treated with G418 (200 µg/ml) for 72 hours. Western blots were probed with the FE-9 antibody directed against the N-terminus of APC. The truncated forms corresponding to the mutated alleles present in LoVo cells are indicated by arrows. An extract from HeLa cells (APC WT) was used as a control; a band was detected at 311 kDa.</p

APC biological activity is correlated with antibiotic-induced readthrough level in human colorectal cancer cells.

<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

APC nonsense mutations.

<p>*Mutations are named by the position and the nature of the wild-type amino acid in APC protein sequence.</p>§<p>These nonsense mutation sequences were inserted into the dual reporter vector in order to determine readthrough level.</p>†<p>Frequencies were given relative to total nonsense mutations listed for APC gene.</p>‡<p>This nonsense mutation was reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024125#pone.0024125-Oh1" target="_blank">[33]</a>.</p

Readthrough levels and factor of increase for 66 sequences containing a stop codon.

<p>Readthrough efficiencies of 66 sequences (65 nonsense mutations and 1 natural stop codon) were measured in NIH3T3 cells with and without gentamicin (800 µg/ml) treatment for 24 H. Each value shown is the mean of at least three independent experiments. For each sequence, the factor of increase (I) is the ratio of the gentamicin-induced readthrough level (G) to the basal readthrough level (B). Sequences are ranked in descending order of gentamicin-induced readthrough level.</p

Effect of stop codon identity on readthrough levels and the factor of increase.

<p>Graphical representation of medians (before Box-Cox transformation) for the parameters B, G and I are shown for the three different stop codons: UAA, UAG and UGA. The results of the statistical analysis after Box-Cox transformation are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002608#pgen.1002608.s008" target="_blank">Table S5</a>. For basal and gentamicin-induced readthrough levels, the following hierarchy of readthrough level was established: UGA>UAG>UAA. (with>indicating a significant difference). For the factor of increase, UGA = UAG = UAA.</p

Statistical approach.

<p>Description of the statistical approach used for the study of the impact of identity of the stop codon or nucleotide context on readthrough levels and response to gentamicin.</p

Effect of nucleotide identity at each position (−6 to +9) on readthrough levels and the factor of increase.

<p>Graphical representations of medians for the parameters B, G and I (before Box-Cox transformation) for the four different nucleotides (A, C, G or U) at each position in the sequence surrounding the codon stop (−6 to +9). Statistical analysis was performed after Box-Cox transformation and results are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002608#pgen-1002608-t001" target="_blank">Table 1</a> and detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002608#pgen.1002608.s009" target="_blank">Table S6</a>.</p

Graphical representations of the distribution of values for the parameters B, G, and I.

<p>For each parameter, values are classified into intervals of equal size. X-values indicate the limits for intervals. Y-values indicate the number of values in each interval.</p

Graphic representations of correlation between the variables B, G, and I after Box-Cox transformation.

<p>Gentamicin-induced readthrough level is plotted against basal readthrough level (A); the factor of increase is plotted against basal readthrough level (B) and against gentamicin-induced readthrough level (C). Bravais-Pearson tests were performed to analyze the correlations between the three variables after Box-Cox transformation. Statistically significant results are indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002608#pgen.1002608.s007" target="_blank">Table S4</a>.</p