Additional file 1 of Umbilical cord mesenchymal stromal cells in serum-free defined medium display an improved safety profile

Additional file 1. Table S1: Flow cytometry antibodies from BD bioscience

Different synMuv B classes distinctly repress P granule and RNAi genes in the soma.

<p>Real-time RT-PCR assays were performed using L4 stage worms with the indicated genotype or RNAi treatment. Expression levels were compared to that of <i>glp-4</i> worms and fold upregulations are plotted on the Y-axis. An asterisk indicates that the synMuv B gene was inactivated by RNAi in <i>glp-4(bn2); eri-1(mg366)</i> double mutant animals and expression levels were compared to that of vector control treated animals. Fold upregulations represent target expression in the soma, except for those in wild type animals, which represent the germline-enriched nature of the target gene. Asterisks represent greater than 2-fold mean fold change and with a <i>p</i>-value of less than 0.05 in two-tailed t-tests. Target genes were categorized into (A) germline-specific common targets, (B) ubiquitously expressed common targets, (C) DRM-specific targets, (D) synMuv B heterochromatin and MEP-1-LET-418 specific targets.</p

A synMuv B heterochromatin complex.

<p>(A) LIN-61::GFP fusion proteins concentrate at nuclear foci, similar to HPL-2::GFP foci, as shown by live fluorescent microscope images and zoomed in images of early embryos. (B) LIN-61::GFP foci fall into regions with low DAPI staining. Early embryos expressing LIN-61::GFP were costained with an anti-GFP antibody and DAPI. The merged image represents a zoom-in of the boxed region in the whole embryo and was contrast enhanced to highlight the localization of concentrated GFP foci in DAPI holes. Arrows point to LIN-61::GFP negative nuclei, which have condensed chromatin and are likely undergoing mitosis. (C) LIN-13::GFP and LIN-61::3xFLAG fusion proteins localize to overlapping nuclear foci. Embryos coexpressing both fusion proteins were costained with anti-GFP and anti-FLAG antibodies. A merged image that zooms into the boxed region in the whole embryo is also shown. (D) LIN-61::GFP interacts with LIN-13 and HPL-2. Anti-GFP immunoprecipitate from LIN-61::GFP expressing embryos was analyzed by mass spectrometry. Peptide coverage data for synMuv B heterochromatin class proteins were shown. (E) LIN-13::GFP and HPL-2::GFP interact with LIN-61::3xFLAG protein in co-IP western assays. Only HPL-2::GFP showed interaction with LIN-54::3xFLAG but the interaction was weaker compared to its interaction with LIN-61::3xFLAG. Each input lane represents 0.4% of material used in IP.</p

DRM and synMuv B heterochromatin class genes provide nonoverlapping functions.

<p>(A) DRM; synMuv B heterochromatin double mutant animals showed further enhanced RNAi efficiency than either single null mutant, as shown by the increased penetrance of RNAi phenotypes in double mutants (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002542#s4" target="_blank">Materials and Methods</a>). (B) Additive upregulation of common targets in <i>glp-4; hpl-2</i> or <i>glp-4 lin-35</i> animals each treated with additional <i>synMuv B(RNAi)</i>. Real-time RT-PCR assays were performed on L4 stage animals and expression levels were each compared to those in respective vector control RNAi treated animals. Asterisks represent a <i>p</i>-value of less than 0.05 in two-tailed t-tests.</p

Different synMuv B classes distinctly repress P granule and RNAi genes in the soma.

<p>Real-time RT-PCR assays were performed using L4 stage worms with the indicated genotype or RNAi treatment. Expression levels were compared to that of <i>glp-4</i> worms and fold upregulations are plotted on the Y-axis. An asterisk indicates that the synMuv B gene was inactivated by RNAi in <i>glp-4(bn2); eri-1(mg366)</i> double mutant animals and expression levels were compared to that of vector control treated animals. Fold upregulations represent target expression in the soma, except for those in wild type animals, which represent the germline-enriched nature of the target gene. Asterisks represent greater than 2-fold mean fold change and with a <i>p</i>-value of less than 0.05 in two-tailed t-tests. Target genes were categorized into (A) germline-specific common targets, (B) ubiquitously expressed common targets, (C) DRM-specific targets, (D) synMuv B heterochromatin and MEP-1-LET-418 specific targets.</p

<i>mes-4/SET histone methyltransferase</i>, a synMuv B suppressor, preferentially counteracts the activity of the synMuv B heterochromatin class proteins.

<p>Real-time RT-PCR assays were performed using L4 stage worms with the indicated genotype and RNAi treatment. Expression levels of a given target gene were compared to that of <i>glp-4</i> worms treated with vector RNAi. Target genes were categorized into (A) germline-specific common targets, (B) ubiquitously expressed common targets, (C) DRM-specific targets, (D) synMuv B heterochromatin and MEP-1-LET-418 specific targets.</p

MEP-1 and LET-418 function as the Mec complex to prevent somatic germline gene expression by mediating the repressive effect of sumoylation.

<p>(A) SUMO, but not NuRD, is required to prevent somatic PGL-1 misexpression. Mixed stage animals with the indicated genotype or RNAi treatment were subjected to anti-PGL-1 immunostaining, and the percentages of animals showing somatic expression were plotted. n equals the total number of animals being assayed. Images of anti-PGL-1 stained animals were shown on the right. Arrows point to misexpressed PGL-1 granules in the intestine. Note the densely clustered small granules in RNAi treated animals. (B) SUMO, but not NuRD, is required to repress other germline-specific targets. Real-time RT-PCR assays were performed using L4 stage <i>glp-4</i> worms with the indicated RNAi treatment. Expression levels were compared to that of <i>glp-4</i> worms treated with control RNAi, and fold upregulations were plotted on the Y-axis. Fold upregulations represent target expression in the soma.</p

Canonical variate analysis (CVA) based on morphological characters of inferred mitochondrial lineages of <i>P. huangchuchieni</i>.

<p>Canonical variate analysis (CVA) based on morphological characters of inferred mitochondrial lineages of <i>P. huangchuchieni</i>.</p

Factor loadings for the 10 highest measured variables and canonical correlations of the differentiation analysis for the 5 main lineages.

<p>Factor loadings for the 10 highest measured variables and canonical correlations of the differentiation analysis for the 5 main lineages.</p

Molecular Phylogeography and Evolutionary History of <i>Poropuntius huangchuchieni</i> (Cyprinidae) in Southwest China

<div><p>Background</p><p>The evolution of the Yunnan Plateau’s drainages network during the Pleistocene was dominated by the intense uplifts of the Qinghai-Tibetan Plateau. In the present study, we investigated the association between the evolutionary histories of three main drainage systems and the geographic patterns of genetic differentiation of <i>Poropuntius huangchuchieni</i>.</p><p>Methodology/Principal Findings</p><p>We sequenced the complete sequences of mitochondrial control region for 304 specimens and the sequences of Cytochrome <i>b</i> gene for 15 specimens of the species <i>P. huangchuchieni</i> and 5 specimens of <i>Poropuntius opisthoptera</i>. Phylogenetic analysis identified five major lineages, of which lineages MK-A and MK-B constrained to the Mekong River System, lineages RL and LX to the Red River System, and lineage SW to the Salween River System. The genetic distance and network analysis detected significant divergences among these lineages. Mismatch distribution analysis implied that the population of <i>P. huangchuchieni</i> underwent demographic stability and the lineage MK-B, sublineages MK-A1 and LX-1 underwent a recent population expansion. The divergence of the 5 major lineages was dated back to 0.73–1.57 MYA.</p><p>Conclusions/Significance</p><p>Our results suggest that <i>P. opisthoptera</i> was a paraphyletic group of <i>P. huangchuchieni</i>. The phylogenetic pattern of <i>P. huangchuchieni</i> was mostly associated with the drainage’s structures and the geomorphological history of the Southwest Yunnan Plateau. Also the differentiation of the major lineages among the three drainages systems coincides with the Kunlun-Yellow River Movement (1.10–0.60 MYA). The genetic differentiation within river basins and recent demographical expansions that occurred in some lineages and sublineages are consistent with the palaeoclimatic oscillations during the Pleistocene. Additionally, our results also suggest that the populations of <i>P. huangchuchieni</i> had keep long term large effective population sizes and demographic stability in the recent evolutionary history, which may be responsible for the high genetic diversity and incomplete lineages sorting of <i>Poropuntius huangchuchieni.</i></p></div