Relative quantification of mtDNA and cell growth analysis of PC-3 fusion cells.

<p>A Analysis of mtDNA relative to nuclear DNA over a period of 18 weeks after cell fusion of PC-3 ρ⁰ cells and cytoplasts of PC-3 MTS-DsRed (F 1–3, solid lines) or PC-3 MTS-EGFP ρ⁰ cells and cytoplasts of PC-3 MTS-DsRed (F A) and cytoplasts of 143B.TK^- MTS-DsRed (F B) (dashed lines) using a primer set for amplification of nuclear (18S rDNA) and mitochondrial (ND5 gene) DNA. Data were normalized to measurements of untreated wild type cells (dotted line). Data represent means ± SD obtained in three determinations. <b>B</b> Calculation of doubling time of PC-3 cells in media with uridine (dark grey bars) and without uridine (light grey bars). Total cell number was measured every 24 h over a period of six days and cell doubling was estimated using exponential regression. The presented data are means ± SD obtained in four experiments. Significance levels between wild type and the different fusion cell lines or if designated between two fusion cell lines were indicated. *P<0.05, **P<0.01, ***P<0.001. <i>B Inset</i> Comparison of relative mtDNA content and cell doubling time. MtDNA content and cell doubling time of PC-3 fusion cells cultured without uridine (close symbols) were normalized to wild type cells and a linear regression was performed (solid line).</p

Polarographic respiration measurements of PC-3 fusion cells.

<p><b>A</b> Endogenous oxygen consumption rate of intact PC-3 fusion cells was measured in TD buffer and normalized to cell number of the sample. <b>B</b> Relative oxygen consumption rates of PC-3 fusion cells were measured in TD buffer in absence and presence of the uncoupler DNP (dark grey bars). COX capacity of PC-3 fusion cells was measured as relative uncoupled respiration rate at 0.4 mM TMPD in antimycin-treated cells (light grey bars). The data represent means ± SD from 4–6 independent experiments. *P<0.05, **P<0.01, ***P<0.001.</p

Primer combinations for amplification of EcoRI gene sequence sections.

<p>Primer combinations for amplification of EcoRI gene sequence sections.</p

Nomenclature of fusion cell lines.

<p>Nomenclature of fusion cell lines.</p

Activity of mitochondrial enzymes in PC-3 fusion cells.

<p>Enzyme activity of mitochondrial citrate synthase, respiratory complex I and IV was measured spectrophotometrically in total cell lysates and normalized to wild type. The data represent means ± SD from four independent experiments. *P<0.05, **P<0.01, ***P<0.001. Specific activity means of mitochondrial enzymes are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073207#pone.0073207.s003" target="_blank">Table S1</a>.</p

Characterization of ρ⁰ cells.

<p>A Multiplex PCR analysis of mitochondrial DNA. The nuclear and mitochondrial genes (18S rDNA: 229 bp and D-Loop: 436 bp, respectively) were coamplified and visualized by gel electrophoresis. Agarose gel (1.5%), lane 1 and 6: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 wild type, lane 3: PC-3 ρ⁰ EtBr, lane 4: PC-3 ρ⁰ 9B4, lane 5: no template. <b>B</b> PCR analysis of EcoRI gene sequence in PC-3 ρ⁰ 9B4 cells. Different EcoRI gene sequences were amplified by PCR from PC-3 ρ⁰ 9B4 genomic DNA utilizing the primer pairs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073207#pone-0073207-t002" target="_blank">Table 2</a>. Agarose gel (1.5%), lane 1 and 17: GeneRuler™ 100 bp Plus DNA Ladder, lane 2–4: PC-3 ρ⁰ 9B4, vector DNA (encoding mitochondrial targeted restriction endonuclease, 500 pg) and no template, primer pair A, lane 5–7: primer pair B, lane 8–10: primer pair C, lane 11–13: primer pair D, lane 14–16: primer pair E. <b>C</b> Cell growth analysis of PC-3 cells. Calculation of doubling time of PC-3 cells in media with uridine (dark grey bars) and without uridine (light grey bars). Total cell number was measured every 24 h over a period of six days and cell doubling was estimated using exponential regression. The presented data are means ± SD from four independent experiments. *P<0.05, **P<0.01, ***P<0.001.</p

PCR analysis of MTS-DsRed gene sequence in nuclear genome.

<p>A 681 bp fragment of the DsRed gene was amplified by PCR as described in the experimental procedures. <b>A</b> Agarose gel (1.5%), lane 1 and 15: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 MTS-DsRed, lane 3: EtBr F 1, lane 4: EtBr F 2, lane 5∶9B4 F 1, lane 6∶9B4 F 2, lane 7: EtBr F 4, lane 8: EtBr F 3, lane 9∶9B4 F 5, lane 10∶9B4 F 4, lane 11∶9B4 F 3, lane 12: PC-3 ρ⁰ EtBr, lane 13: PC-3 ρ⁰ 9B4, lane 14: no template control. <b>B</b> Agarose gel (1.5%), lane 1 and 10: GeneRuler™ 100 bp plus DNA Ladder, lane 2∶143B.TK^- MTS-DsRed, lane 3: PC-3 MTS-EGFP ρ⁰ EtBr, lane 4: PC-3 MTS-EGFP ρ⁰ 9B4, lane 5: EtBr F B, lane 6∶9B4 F B, lane 7: EtBr F A, lane 8∶9B4 F A lane 9: no template control.</p

Confocal images of ρ⁰ cells fused with cytoplasts of wild type cells.

<p>PC-3 ρ⁰ 9B4 cells stably expressing mitochondrial targeted EGFP (MTS-EGFP) were fused with cytoplasts of PC-3 (panel A) or 143B.TK^- (panel B) stably expressing mitochondrial targeted DsRed (MTS-DsRed) and were analyzed after 24 h by confocal laser scanning microscopy. Scale bars correspond to 10 µm.</p

Distribution of <i>CEL</i> VNTR lengths for alcoholic chronic pancreatitis (ACP) and alcoholic liver cirrhosis (ALC) patients from Germany.

<p>Distribution of <i>CEL</i> VNTR lengths for alcoholic chronic pancreatitis (ACP) and alcoholic liver cirrhosis (ALC) patients from Germany.</p

Genotype distribution of <i>CEL</i> VNTR lengths for alcoholic liver cirrhosis (ALC) patients and controls from the United Kingdom.

<p>Genotype distribution of <i>CEL</i> VNTR lengths for alcoholic liver cirrhosis (ALC) patients and controls from the United Kingdom.</p