Influence of fluorescent tags on MMR activity of MutLα.

<p>HEK293T cells were transiently cotransfected with various labeled or unlabeld MutLα constructs and 48 h post transfection MMR activity of different MutLαs were assessed in vitro in parallel with unlabeled MutLα by quantifying the 3′-nick-directed correction of a G-T mismatch in a restriction site of a plasmid substrate as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#s4" target="_blank">Materials and Methods</a>” and as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#pone.0031863-Brieger3" target="_blank">[10]</a>. <i>In vitro</i> repair was scored on a 2-kb circular DNA substrate that contains an <i>EcoRV</i> site which is destroyed by a G-T mismatch. Upon repair of the G-T mismatch to an A-T base pair the intact <i>EcoRV</i> site together with an <i>AseI</i> site gives rise to a 0.8- and a 1.2-kb fragment, whereas unrepaired DNA is only linearized by <i>AseI</i> to a 2-kb fragment. Repair efficiency was assessed by measuring the signal intensities of linearized and digested vector with Bio-Rad Quantity One software using the “rolling ball” baseline correction. The signal intensity of the repair bands was divided by the intensity of all three bands. Repair efficiency of unlabeled MutLα was set at 100 percent and repair of fluorescent tagged MutLα was determined in relation to the wild-type sample that was expressed, processed and tested in parallel. Average repair values and standard deviations (±) were determined from four independent experiments. Single PMS2 tagged MutLαs, single MLH1-GFP-N tagged MutLαs as well as MLH1-GFP-N coexpressed with PMS2-Red-N or MLH1-Red-N coexpressed with PMS2-GFP-N were MMR proficient while all other tagged variants showed MMR deficiency. 1: mock control (untransfected). 2: MLH1/PMS2 unlabeled (positive control); 3: MLH1/PMS2-GFP-N; 4: MLH1/PMS2-GFP-C; 5: MLH1/PMS2-Red-N; 6: MLH1/PMS2-Red-C; 7: MLH1-GFP-N/PMS2; 8: MLH1-GFP-C/PMS2; 9: MLH1-Red-N/PMS2; 10: MLH1-Red-C/PMS2; 11: MLH1-GFP-N/PMS2-Red-N; 12: MLH1-GFP-N/PMS2-Red-C; 13: MLH1-GFP-C/PMS2-Red-N; 14: MLH1-GFP-C/PMS2-Red-C; 15: MLH1-Red-N/PMS2-GFP-N; 16: MLH1-Red-N/PMS2-GFP-C; 17: MLH1-Red-C/PMS2-GFP-N; 18: MLH1-Red-C/PMS2-GFP-C. Symbols see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#pone-0031863-g001" target="_blank">Figure 1</a>.</p

Dye tags influence single expression of MLH1 and PMS2.

<p>To determine the influence of fluorescent tags on single expressed MLH1 or PMS2 variants, HEK293T cells were transfected with different (A) MLH1 or (B) PMS2 constructs. Amounts of expressed proteins were assessed after Western blotting by measuring the signal intensities of protein bands with Multi Gauge V3.2 software. Graphs indicate the results (mean ±S.D.) of at least four independent experiments in which the proportion of protein expression using an unbiased method were presented. 1: MLH1 unlabeled; 2: MLH1-GFP-N; 3: MLH1-GFP-C; 4: MLH1-Red-N; 5: MLH1-Red-C; 6: PMS2 unlabeled; 7: PMS2-GFP-N; 8: PMS2-GFP-C; 9: PMS2-Red-N; 10: PMS2-Red-C.</p

Putative three-dimensional structure models of fluorescent labeled MutLα.

<p>Using PyMol (Warren DeLano, <a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>), GFP or Red fluorescent proteins were attached to (A) N-termini of MLH1 and PMS2 or (B) C-termini of MutLα. C-terminal tags seem to hide the C-terminal region of MutLα and consequently might avoid DNA interaction. (C) Corresponding amino acid sequences of linker regions are shown. The dashed line between PMS2 and the N-terminal fluorescent tag illustrates a putative α-helix (unknown structure) of the first thirty amino acids of PMS2.</p

Influence of fluorescent labeling of MutLα on protein expression levels.

<p>To analyze the effect of fluorescent dyes on protein expression levels, HEK293T cells were transiently cotransfected with different MutLα constructs (see below) and (A) Western blot analysis was carried out after 48 h using anti-MLH1 or anti-PMS2, respectively, controlled by β-actin detection. (B) Amounts of expressed proteins were assessed by measuring the signal intensities of protein bands with Multi Gauge V3.2 software. Graphs indicate the results (mean ±S.D.) of at least four independent experiments in which the proportion of protein expression using an unbiased method were presented. 1: negative control (untransfected). 2: MLH1/PMS2 unlabeled; 3: MLH1/PMS2-GFP-N; 4: MLH1/PMS2-GFP-C; 5: MLH1/PMS2-Red-N; 6: MLH1/PMS2-Red-C; 7: MLH1-GFP-N/PMS2; 8: MLH1-GFP-C/PMS2; 9: MLH1-Red-N/PMS2; 10: MLH1-Red-C/PMS2; 11: MLH1-GFP-N/PMS2-Red-N; 12: MLH1-GFP-N/PMS2-Red-C; 13: MLH1-GFP-C/PMS2-Red-N; 14: MLH1-GFP-C/PMS2-Red-C; 15: MLH1-Red-N/PMS2-GFP-N; 16: MLH1-Red-N/PMS2-GFP-C; 17: MLH1-Red-C/PMS2-GFP-N; 18: MLH1-Red-C/PMS2-GFP-C. Symbols see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#pone-0031863-g001" target="_blank">Figure 1</a>.</p

MSP analysis.

<p>Representative examples of MSP analysis for MLH1, MSH2, PMS2 and p16 methylation in HCC and non-tumour adjacent tissues were shown. Bisulfite-modified DNA was amplified using MSP primers specific to a CpG-rich region of each gene promoter. PCR-amplified products were resolved by 2% agarose gel electrophoresis. (U) Lanes represent amplification of unmethylated alleles, and (M) lanes contain only methylated alleles.</p

Frequency of promoter methylation.

<p>Frequency of promoter methylation.</p

Clinical feature of the studied patients.

<p>Clinical feature of the studied patients.</p

MSI analysis.

<p>Analysis of commonly used mononucleotide MSI marker loci BAT25 and BAT26 was performed in all patients showing promoter methylation in one of the tested MMR genes. Three examples are shown here. (T) HCC tissue, (NT) non-tumour adjacent tissue.</p

Primer sequences and annealing temperatures used in MSP.

<p>Primer sequences and annealing temperatures used in MSP.</p

Relationship between promoter methylation and clinicopathologic parameters of HCC patients (N = 61).

<p>Relationship between promoter methylation and clinicopathologic parameters of HCC patients (N = 61).</p