Conservation of microRNA target sites in selected amniotes and the presence in <i>Twist2</i> 3′UTR.

<p>MicroRNAs underlined were tested in this study.</p

MicroRNA target sites are necessary for the repression of <i>Twist1</i> 3′UTR reporter.

<p>MiRNAs of interest were co-transfected with either wt or the corresponding mutant of psiCheck2-Twist1-3′UTR reporter into H1299 cells. Firefly luciferase activities were measured after 48 h, normalized to <i>Renilla</i> luciferase activities and subsequently to the respective wt+miRNA. Statistical significance was calculated by using Student's t-test and by comparing the mutant 3′UTR values with the wt 3′UTR reporter.</p

MicroRNAs repress the <i>Twist1</i> 3′UTR reporter.

<p>H1299 cells were transfected with the indicated miRNA, pRluc-N2 and pGL3-Twist1-3′UTR Firefly luciferase reporter or the empty pGL3-control vector and analyzed after 48 h. Firefly luciferase activities were normalized to the <i>Renilla</i> luciferase activities which served as internal standards, averages of triplicates were calculated and results were normalized to empty pGL3-control vector. The dashed line indicates the unrepressed expression level of the reporter (0,176; calculated from the average of two negative controls (*), miR-485 and miR-609). Statistical significance of miRNA effects was calculated by comparison with this average using Student's t-test.</p

MicroRNAs 151-5p and 337-3p reduce the mobility of murine embryonic fibroblast cells.

<p>Dot-plot superposed with the box-plot of the predicted probabilities of cell migration for observations from miR-151-5p + miR-337-3p (red) and scrambled control (blue) treated cells (<i>p</i>-value 0.000135). Note the presence of miR-151-5p + miR-337-3p treated cells with extreme low estimates of probability of cell migration.</p

Endogenous microRNAs reduce the activity of <i>Twist1</i> 3′UTR reporter.

<p>NIH-3T3 (A) and C3H/10T1/2 (B) cells were transfected with wt or mutant psiCheck2-Twist1-3′UTR reporter only. Endogenous miRNAs in NIH-3T3 (C) and C3H/10T1/2 (D) cells were inhibited by co-transfection of 50 nM miRCURY LNA microRNA inhibitors and wt psiCheck2-Twist1-3′UTR reporter. Firefly luciferase activities were measured after 48 h, normalized to <i>Renilla</i> luciferase activities and subsequently to the wt 3′UTR (A-B) or wt+anti-Scrambled (C-D). Statistical significance was calculated by using Student's t-test and by comparing the mutant <i>Twist1</i> 3′UTR reporters or miRNA specific antagonists with the wt 3′UTR reporter or a scrambled control (*), respectively.</p

Pairwise comparison of <i>Twist1</i> and <i>Twist2</i> mRNA sequence domains of three selected amniotes with the corresponding human sequence. Numbers represent% sequence identity.

1<p>CDS: coding sequence; ²UTR: untranslated region.</p

MicroRNAs lead to translational inhibition of <i>Twist1</i> 3′UTR reporter.

<p>(A) H1299 cells were co-transfected with wt psiCheck2-Twist1-3′UTR reporter and 20 nM synthetic precursor miRNAs. (B) H1299 cells stably expressing the psiCheck2-Twist1-3′UTR reporter were transfected with 5 nM synthetic precursor miRNAs. <i>Renilla</i> luciferase activity was measured after 48 h and normalized to the Firefly luciferase activity. <i>Renilla</i> luciferase mRNA levels were measured by qPCR, normalized to Firefly luciferase mRNA levels and subsequently to cells transfected with the negative control.</p

MicroRNA expression levels in different mouse cell lines.

<p>Endogenous miRNA expression levels in NIH-3T3, C3H/10T1/2 and C₂C₁₂ cells were measured by qPCR using TaqMan probes, and normalized to U6 snRNA. Due to the high expression of miR-145a-5p, the relative values are presented on a logarithmic scale.</p

Transposon copy number in the RMCE-in HEK, HeLa, and murine ES cell clones.

<p><b>(A)</b> Southern blot of selected genomic DNA from <i>KpnI</i> digested HEK, (<b>B)</b> HeLa and <i>PstI</i> digested (<b>C)</b> mES clones. A 700bp dCTP³² labeled RFP probe (red rectangle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g001" target="_blank">Fig 1A</a>) identified transposition of the gene cassette at bands > 2900bp as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g001" target="_blank">Fig 1A</a>. Clone numbers are depicted above each lane.</p

Design of the RMCE docking site.

<p>(A) <i>Left</i>: Schematics of the commercially available Flp-in docking site. <i>Right</i>: Design of the SB transposon constituting the RMCE docking site present in the new RMCE-in cell lines. The RMCE docking site contains the CAG promoter which drives the expression of a RFP reporter linked to the puromycin-resistant gene through the ribosomal skip element E2A. (B) Schematic representation of donor plasmid used for Flp-in (left) and RMCE-in (right). The genetic element of interest (GEI) is represented by a GFP reporter. Note that the GFP in the RMCE-in donor does not contain a poly(A)-signal and utilizes the poly(A) from the RMCE docking site. The RMCE-in and the Flp-in donor plasmid are compatible with both the RMCE-in and Flp-in cell line. (C) Post recombination of the Flp-in system <i>left</i>: Prokaryotic elements, the initial marker and selection gene are present in the commercial Flp-in^™-293 cells post recombination, while the RMCE-in <i>right</i> leaves no prokaryotic DNA or initial reporter genes after cassette exchange.</p