() MDS cells are mosaic for expression of mtDNA and mitochondrial transcription factor A (TFAM) and have reduced mtDNA synthesis compared with controls

(a) Control fibroblasts co-labelled with anti-DNA antibody (green) and Mitotracker red, showing orange-yellow labelling were there two signals co-localize. (b) Patient A fibroblasts co-labelled with anti-DNA/Mitotracker. (DAPI was used to visualize nuclei blue and an asterisk denotes a cell with no anti-DNA signal). (c) Patient B fibroblasts co-labelled with anti-DNA/Mitotracker. (note: a reduced anti-DNA signal is shown by the predominantly red co-localization with Mitotracker). (d) Patient C fibroblasts co-labelled with anti-DNA/Mitotracker (arrow shows area of residual anti-DNA/mitotracker co-labelling within a depleted cell with reduced Mitotracker labelling). (e) Control fibroblasts co-labelled with anti-TFAM antibody (green) and Mitotracker red. (f) Patient A fibroblasts co-labelled with anti-TFAM/Mitotracker (asterisks show TFAM depleted cells with reduced Mitotracker labelling). (g) Patient B fibroblasts co-labelled with anti-TFAM/Mitotracker. (h) Patient C fibroblasts co-labelled with anti-TFAM/Mitotracker. (i–l) Fibroblasts pulsed with Bromodeoxyuridine (BrdU) for 220 min, and Br-DNA immuno-detected. (i) BrdU labelling of normal fibroblasts, (j) BrdU labelling of patient A cells (inset shows magnified area of cytoplasm showing weak BrdU labelling). (k) BrdU labelling of patient B cells. (l) BrdU labelling of patient C cells (note: asterisks mark ‘ρ° type’ cells with very little or no mtDNA labelling). Bar 20 µM. () Expression of mtDNA encoded cytochrome oxidase subunit I (COXI) in MDS patients is reduced, as is COX activity, but not SDH activity. Cytochrome oxidase subunit I (COXI) expression was monitored using immuno-cytochemistry. (a) COXI labelling of normal control fibroblasts. (b) COXI labelling of patient A fibroblasts (COXI depleted cells are asterisked). (c) COXI labelling of patient B fibroblasts. (d) COXI labelling of patient C fibroblasts. (e–h) Histochemical demonstration of COX activity in fibroblasts. (e) COX activity of control. (f) COX activity of patient A cells (asterisks denote cells with markedly reduced activity). (g) COX activity of patient B cells. (h) COX activity of patient C cells. (i–l) Histochemical demonstration of SDH activity in fibroblasts. (i) SDH activity of control cells. (j) SDH activity of patient A cells. (k) SDH activity of patient B cells. (l) SDH activity of patient C cells. Bars 20 µM. () Tetramethyl-rhodamine-methyl ester (TMRM)/PicoGreen co-labelling of mosaic MDS fibroblast mitochondrial membrane potential. (a) PicoGreen labelling of normal control fibroblasts. (b) Co-staining of the same cells with TMRM. (c) Co-localization of the two signals in (a) and (b). (d and e) PicoGreen/TMRM co-labelling of later passage Patient A's fibroblasts. (f) Co-localization of the two signals in (d) and (e). (g and h) PicoGreen/TMRM co-labelling of later passage Patient B's fibroblasts. (i) Co-localization of the two signals. (j and k) PicoGreen/TMRM co-staining of late passage Patient C's cells. (l) Co-localization of the two signals. (m and n) PicoGreen/TMRM co-staining of late passage Patient D's cells. (o) Co-localization of the two signals. Bars 20 µm.<p><b>Copyright information:</b></p><p>Taken from "Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations"</p><p></p><p>Human Molecular Genetics 2008;17(16):2496-2506.</p><p>Published online 16 May 2008</p><p>PMCID:PMC2486441.</p><p>© 2008 The Author(s)</p

() Quantification of PicoGreen staining of proportion of mosaic MDS cell cultures A–C that appeared depleted over time

The number of cells that appeared depleted of mtDNA increased over 45 days in the patients but not the controls (200 cells were counted at each time point). Numbers were significantly higher in patients than controls at all time points, other than 28 days (0.05< < 0.0016). () Quantification of the numbers of nucleoids visible by PicoGreen staining of mosaic MDS cell cultures A–C and controls [same experiment as () and ]. Nucleoid numbers drop with time in MDS cultures ( < 0.001 and <p><b>Copyright information:</b></p><p>Taken from "Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations"</p><p></p><p>Human Molecular Genetics 2008;17(16):2496-2506.</p><p>Published online 16 May 2008</p><p>PMCID:PMC2486441.</p><p>© 2008 The Author(s)</p