Loss of MSH6 reduces the frequency of paternally and maternally transmitted expansions.

<p>The % of alleles with the indicated number of repeats added or lost seen on transmission of a PM allele with ~160 repeats from <i>Msh6</i>^<i>+/+</i>, <i>Msh6</i>^+/- and <i>Msh6</i>^<i>-/-</i> parents. A total of 44 <i>Msh6</i>^<i>+/+</i>, 70 Msh6^+/- and 41 <i>Msh6</i>^<i>-/-</i> paternal transmissions and 53 <i>Msh6</i>^<i>+/+</i>, 65 <i>Msh6</i>^+/- and 43 <i>Msh6</i>^<i>-/-</i> maternal transmissions were examined. Differences in frequency of expansions, contractions and unchanged alleles were compared for each pair of genotypes using Fisher’s exact test in which each allele class was considered in relation to the sum of the other two allele size classes. The allele classes that are significantly different from <i>Msh6</i>^<i>+/+</i> animals are marked with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001); frequencies were also compared across all 3 genotypes with exact Jonckheere-Terpstra tests (p<0.001 for each sex).</p

Loss of MSH6 reduces somatic expansions in both males and females.

<p>A) Representative examples of the GeneMapper profiles for the PM allele in different organs of 6 month old <i>Msh6</i>^<i>+/+</i> and <i>Msh6</i>^<i>-/-</i> males. Tail 1 refers to the tail DNA at 3 weeks of age, while tail 2 refers to the tail sample taken at 6 months of age. B) The SII for the organs of six 6-month old <i>Msh6</i>^<i>-/-</i> males and six <i>Msh6</i>^<i>+/+</i> littermates and three 12 month old <i>Msh6</i>^<i>-/-</i> females and four <i>Msh6</i>^<i>+/+</i> littermates was determined and the average SII for each organ plotted. The initial repeat number in these mice was ~160. The negative value for the heart in animals of each genotype and for the remaining organs in <i>Msh6</i>^<i>-/-</i> mice reflects the strand-slippage or stutter products that are typically seen when large repeat tracts are amplified rather than contraction events. Note that despite the fact that the females are twice as old as the males, the SIIs for all organs in females were either similar to or lower than they were in males. The error bars represent standard deviations.</p

Loss of MSH6 reduces the frequency of expansions but does not significantly reduce the frequency of large contractions in the sperm of <i>Msh6</i>^<i>-/-</i> males.

<p>Small pool PCR was carried out on the sperm of 2-month old males as described in the Materials and Methods. A) Representative GeneMapper profiles for sperm samples analyzed by small-pool PCR showing the original paternal allele and 5 alleles, labeled S1-S5, found in sperm. B) The % of alleles with the loss or gain of different repeat numbers seen in the sperm of <i>Msh6</i>^<i>+/+</i> and <i>Msh6</i>^<i>-/-</i> mice. A total of 135 and 173 alleles were examined in the sperm of <i>Msh6</i>^<i>+/+</i> and <i>Msh6</i>^<i>-/-</i> mice respectively. The proportion of contractions, expansions and alleles that are the same size as the parental allele are indicated above each graph. Differences between these allele classes were compared for each pair of genotypes using Fisher’s exact test in which each allele class was considered in relation to the sum of the other two allele size classes. The allele classes that are significantly different from <i>Msh6</i>^<i>+/+</i> animals are marked with asterisks (p<0.0001). Note that the proportion of the different allele classes differs from the data shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006190#pgen.1006190.g002" target="_blank">Fig 2</a>. This difference is likely due to the differences in the ages of the animals in the two data sets since in males the number of expansions increases with age, although it is also possible that this reflects, in part, the contribution of other cell types that might be present in the sperm sample. Note that, in contrast to what is seen in <i>Msh3</i>^<i>-/-</i> animals, the distributions of the contracted alleles for both genotypes significantly deviated from unimodality (Hartigans’ dip test: p < 2.2e-16).</p

MutSα binds to both strands of the FX repeat and promotes binding of MutSβ.

<p>MutSα and/or MutSβ with the N-terminal truncation of MSH3 were added at the indicated concentrations to reaction mixtures containing the CCG repeat and CGG repeat-containing substrates as described in the Materials and Methods. The DNA and DNA:protein complexes were then resolved by native polyacrylamide gel electrophoresis at 4˚C, transferred to nylon membrane and the DNA detected using streptavidin conjugated to horseradish peroxidase (HRP) and a chemiluminescent substrate. The different DNA:MutS complexes are numbered in order of decreasing mobility. The arrowheads indicate novel shifted bands that are not seen in reactions containing either MutSα or MutSβ alone.</p

Comparison of free energy at 37˚C and enthalpy changes required to melt the secondary structure formed by CCG-repeats in the presence of BSA and MutSα.

<p>Comparison of free energy at 37˚C and enthalpy changes required to melt the secondary structure formed by CCG-repeats in the presence of BSA and MutSα.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

The loss of MSH6 does not affect the levels of MSH3 in brain and testes.

<p>Equivalent amounts of total protein from brain, testes and ovary and nuclear extracts from liver of three 6-month old WT, <i>Msh3</i>^<i>-/-</i> and <i>Msh6</i>^<i>-/-</i> and <i>Msh2</i>^<i>-/-</i> mice were subjected to electrophoresis and western blotting using the MSH2, MSH3 and MSH6 antibodies described in the Materials and Methods. Nuclear extracts of liver were used to enrich for the MMR proteins that are particularly low in this organ, and to remove a non-specific protein that cross-reacts with the MSH6 antibody. Note that the gel used for nuclear extracts (4–12% Tris-Bis) was different from the gels used to resolve total protein extracts (3–8% Tris-acetate). A representative example of a full blot showing the binding of antibodies to MSH2, MSH3, MSH6 is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006190#pgen.1006190.s001" target="_blank">S1 Fig</a>. Note that in ovary and liver more than one MSH3-species was seen in both WT and <i>Msh6</i>^-/- animals. This most likely reflects proteolytic degradation of MSH3 during preparation of the protein extracts.</p

Additional file 5: Dataset S4. of A next generation sequencing based approach to identify extracellular vesicle mediated mRNA transfers between cells

Differential genes between adipocyte cultured alone and co-cultured with macrophage and between macrophage alone and co-cultured with adipocyte. (XLSX 96 kb

Additional file 1: of A next generation sequencing based approach to identify extracellular vesicle mediated mRNA transfers between cells

A next generation sequencing based approach to identify extracellular vesicle mediated mRNA transfers between cells. (DOCX 1282 kb

Additional file 3: Dataset S2. of A next generation sequencing based approach to identify extracellular vesicle mediated mRNA transfers between cells

Details of mRNA transfer analysis from macrophage to adipocyte. (XLSX 1903 kb