<i>Tol2</i> knockdown vectors were genomically integrated and the inducible expression was tightly controlled <i>in vivo</i>.

<p>(A) Representative neocortical sections showing the basal expression and induced expression of EGFP derived from the pT2K-BI-shRNAmir or pT2K-TBI-shRNAmir vectors. (B) The retention and expression of inducible knockdown vector in glial cells. In the presence of <i>Tol2</i> transposase, EGFP was observed in the glial cells (arrowheads). The right panels are higher magnification views of the boxed regions in the left panels. (C) Representative neocortical sections showing the retention and expression of the inducible knockdown vector in the adult cortex. CP, cortical plate; VZ, ventricular zone. Scale bars, 100 µm.</p

The <i>Tol2</i> transposable vector enables inducible knockdown from a stably integrated knockdown cassette.

<p>(A) A schematic diagram of the pT2K-TBI-shRNAmir vector. The shRNAmir cassette was inserted into the pT2K-BI-TRE-EGFP vector, and TRE-BI was replaced with TRE-TBI. The shRNAmir cassette consisted of the hairpin stem, which is composed of siRNA sense and antisense strands designed for the knockdown of the target gene, a loop derived from human mir30, and mir30 flanking sequences on the 3′ and 5′ sides of the hairpin. (B) A schematic diagram showing the principle of induction of knockdown from the genomically integrated shRNAmir cassette. The <i>Tol2</i>-flanked region of the plasmids were excised and integrated into the chromosome using <i>Tol2</i> transposase. In the presence of Doxycycline (Dox), rtTA-M2 bound to TRE-TBI, and the expression of both EGFP and the mir30-based knockdown cassette were induced under the control of TRE-TBI. (C) Expression of EGFP, induced from the each of pT2K-TBI-shRNAmir vectors (mir-empty, mir-APP#2 and mir-APP#3), was observed in almost all PC12 cells following Dox administration. The upper panels show the bright-field images. Scale bar, 100 µm. (D) Immunoblot analyses for evaluating the knockdown efficiency against APP. Actin was used as a loading control. (E) The basal expression (−Dox) and the induced expression (+Dox) of EGFP from the pT2K-BI-shRNAmir and pT2K-TBI-shRNAmir vectors in HEK293T cells. pCAGGS-tdTomato was co-transfected as a transfection control. Inset shows a higher magnification. Scale bars: 100 µm, inset 20 µm. (F) Ratio of the number of EGFP-positive cells to tdTomato-positive cells between the cells expressing pT2K-BI-shRNAmir and pT2K-TBI-shRNAmir with or without Dox. (mean ± SEM, n = 3). Abbreviations of the vector name and their components are listed in the table (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033380#pone.0033380.s002" target="_blank">Table S1</a>).</p

Univariate analysis of the effect of somatic mutations in the D-loop of mitochondrial DNA on survival of oral squamous cell carcinoma patients.

<p>Univariate analysis of the effect of somatic mutations in the D-loop of mitochondrial DNA on survival of oral squamous cell carcinoma patients.</p

Somatic mutations in the D-loop region of mtDNA of oral cancers.

<p>Abbreviation: mtDNA → mitochondrial DNA</p><p>Somatic mutations in the D-loop region of mtDNA of oral cancers.</p

Disease-specific survival curves of oral squamous cell carcinoma patients based on the somatic mutations in the D-loop of mitochondrial DNA and stratified by pathological stage.

<p>Disease-specific survival curves of oral squamous cell carcinoma patients based on the somatic mutations in the D-loop of mitochondrial DNA and stratified by pathological stage.</p