Additional file 5: of Systems analysis identifies miR-29b regulation of invasiveness in melanoma

Supporting Experimental Data (.pdf) – Contains: Table AF5.1, which lists the PCR primers used. Figure AF5.1 which displays baseline expression for genes of interest in a subset of LM-MEL melanoma cell lines using qPCR. Figure AF5.2, which displays siRNA transfection efficiency; Figure AF5.3, which displays additional qPCR results showing the effects of miR-29b mimic/inhibitor transfection. Figure AF5.4 & AF5.5, which display the effects of miR-29b mimic/inhibitor transfection on LM-MEL-77 cell proliferation and outgrowth. Figure AF5.6, which displays information to aid the reader’s interpretation of melanoma spheroid data. (PDF 3182 kb

Additional file 6: of Systems analysis identifies miR-29b regulation of invasiveness in melanoma

Table (.csv) of Gene Ontology (GO) database terms used for the enrichment analysis of ‘epithelial-mesenchymal plasticity’ (EMP) and pigmentation processes in Fig. 3a, together with the number of genes within each category. As detailed in Methods/Databases, terms were identified by string matching and GO terms were excluded if they had less than five or more than 500 member genes. (CSV 5 kb

Validation of comparative RNA-Seq analysis.

<p>(A) qRT-pCR analysis of representative genes identified by RNA-Seq. Values represent the average transcript copy numbers for each gene normalized per <i>lipL32</i> transcript. Bars indicate the standard error of the mean (SEM). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#s2" target="_blank">Results</a> presented are mean values from at least 3 biologically-independent samples of leptospires for each growth condition. The fold-regulation for each gene determined by RNA-Seq is indicated in parentheses. The fold-regulation between <i>in vitro</i>- (IV) and DMC-cultivated leptospires determined by qRT-PCR are indicated. P values were calculated using an unpaired <i>t</i>-test. (B) Correlation coefficient (R²) between RNA-Seq and qRT-PCR data.</p

Conservation of <i>L. interrogans</i> sv. Copenhageni Fiocruz L1-130 differentially-expressed genes among virulent and saprophytic <i>Leptospira</i> spp. Protein sequence similarities were determined using GLSEARCH (v. 34.05).

<p>Genomes used for analysis: <i>L. interrogans</i> sv. Lai strain 56601, <i>L. borgpetersenii</i> sv. Hardjo strain L550, <i>L. santarosai</i> sv. Shermani strain LT821; <i>L. licerasiae</i> sv. Varillal strain VAR010; and <i>L. biflexa</i> sv. Patoc strain Patoc1 Ames, respectively. The color coding used in the heat map is as follows: blue, 95–100% identity; green, 90–94% identity; orange, 85–89%; and yellow, 80–84%.</p

<i>L. interrogans</i> sv. Copenhageni genes upregulated in DMCs compared to <i>in vitro</i>.

1<p>Gene designations and protein product descriptions are based on those of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento2" target="_blank">[12]</a> and the <i>L. interrogans</i> sv. Copenhageni Genome Project database (<a href="http://aeg.lbi.ic.unicamp.br/world/lic/" target="_blank">http://aeg.lbi.ic.unicamp.br/world/lic/</a>), except where indicated.</p>2<p>nnotation based on Setubal <i>et al.</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Setubal1" target="_blank">[47]</a>.</p>3<p>Revised annotation based on bioinformatics.</p

Summary of RNA-Seq mapping data.

1<p>Total number and percentage (in parenthesis) of reads that mapped to the reference genome with 100% accuracy.</p>2<p>Total number and percentage (in parenthesis) of reads that mapped to a single location within the reference genome <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento2" target="_blank">[12]</a>.</p>3<p>Based on the total of uniquely mapped reads for the corresponding sample.</p

Functional categories of genes differentially-expressed by <i>L. interrogans</i> sv Copenhageni strain Fiocruz L1-130 within DMCs.

<p>Functional categories are based on those of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento2" target="_blank">[12]</a> and the <i>Leptospira interrogans</i> sv. Copenhageni Genome Project database (<a href="http://aeg.lbi.ic.unicamp.br/world/lic/" target="_blank">http://aeg.lbi.ic.unicamp.br/world/lic/</a>). The number of upregulated (Ups) and downregulated (Down) genes within each category are indicated in red and blue, respectively.</p

Candidate small non-coding RNAs identified by RNA-Seq.

1<p>Predicted sRNAs are annotated according to the genome of <i>L. interrogans</i> sv. Copenhageni (LIC) chromosome number followed by non-coding RNA designation as included in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004.s006" target="_blank">Table S2</a>.</p>2<p>Homology to known sRNA families is indicated as is the E-value when transcripts were searched against the Rfam database.</p>3<p>Expression was validated by reverse-transcriptase PCR in <i>L. interrogans</i> sv. Copenhageni strain RJ16441.</p

Mapping of RNA-Seq reads.

<p>Percentage of uniquely mapping reads from each biological replicate of leptospires cultivated in DMCs or under standard <i>in vitro</i> growth conditions (30°C in EMJH).</p

Leptospiral genes differentially-expressed within DMCs compared to <i>in vitro</i>.

1<p>Percentage of genes based on the total number of genes in upregulated or downregulated category.</p>2<p>Percentage of genes based on the total number of differentially-expressed genes.</p>3<p>Hypothetical proteins and uncharacterized lipoproteins.</p