Fig 1.
Loss of mtDNA after induction of the expression of a mitochondrial targeted PstI.
A. Map of the human mtDNA indicating the positions of the unique BamHI site and the two PstI sites, the distances between these sites and the positions of the probes used for the Southern blots (green asterisks). B. Southern blot analysis of the control HEK293 cells (WT) and stably transfected cells (PstI cells) after digestion by BamHI or BamHI + PstI for the control. PstI expression was induced for 2h with doxycycline and the cells were followed during the recovery period, from 0h to 2 days after induction. C. Quantification of the mtDNA/nDNA ratios in the PstI stably transfected cells, without induction or from 0h to 3 days after a 2h induction with doxycycline (mean ± s.e., N = 3). D. Changes in mitochondrial proteins levels during the recovery period after induction of PstI for 2h with doxycycline. Total cell lysates (20 μg) were analyzed by western blot with antibodies against mitochondrial (CO2: complex IV) or nuclear encoded subunits of the respiratory chain (ATP5a: complex V; UQCR2: complex III, SDHB: complex II; NDUFB8: complex I) and against DNA maintenance proteins, the helicase Twinkle, the DNA polymerase POLGA and TFAM. H3: Histone H3B (loading control).
Fig 2.
Effect of silencing different mitochondrial DNA nucleases on the rapid mtDNA depletion.
A. Levels of different mitochondrial DNA nucleases before induction and during the recovery period after induction of PstI for 2h with doxycycline. Total cell lysates (20 μg) were analyzed by western blot with antibodies against ExoG, EndoG, MGME1, and FEN1. H3: Histone H3B (loading control). B. Southern blot analysis of the control HEK293 cells (WT) and stably transfected cells (PstI cells) after digestion by BamHI or BamHI + PstI for the control. The cells were grown in presence or absence of a mix of siRNAs targeting ExoG and/or EndoG. Three days after siRNA transfection, doxycycline was added to the cells for 2h to induce PstI expression, and the cell’s DNA was examined before induction and during the recovery period, at 0h, 5h and 1 day after induction. C, D, E. Similar experiments as in 2B, except that the siRNAs were targeting MGME1, DNA2 or FEN1, respectively.
Fig 3.
The rapid loss of mtDNA is not correlated to a loss of mitochondria, autophagy or apoptosis.
Levels of A. mitochondrial proteins and C. an autophagy marker before induction and during the recovery period after induction of PstI for 2h with doxycycline. Total cell lysates (20 μg) were analyzed by western blot with antibodies against markers of the mitochondrial matrix (PDH), mitochondrial outer membrane (Tomm20), autophagy (LC3) and mitophagy (PINK1). CCCP is used as a positive control and Tubulin is used as loading control. Analysis of B. cell mitochondrial content and D. apoptosis by flow cytometry before induction and during the recovery period. * P ≤ 0.05 versus non-induced PstI cells for each cell population (Student’s t-test).
Fig 4.
The loss of mtDNA is followed by a slow repopulation with intact mtDNA molecules.
A. Southern blot analysis of the control HEK293 cells (WT) and stably transfected cells (PstI cells) after digestion by BamHI or BamHI + PstI for the control. PstI expression was induced for 2h with doxycycline and cells were followed for 20 days. At day 20, a second induction of 2h with doxycycline was performed and the cells were followed during the second recovery period (marked with an apostrophe). B. Quantification of the mtDNA/nDNA ratios in the PstI stably transfected cells, from 0h to 41 days after a 2h induction with doxycycline and from 0h to 20days after a second 2h doxycycline induction. (mean ± s.e., N = 3). C. Quantification of the level of PstI transcripts in the control HEK293 cells (WT) and stably transfected cells (PstI cells), during 2 sequential inductions of PstI expression with doxycycline (mean ± s.e., N = 3). TBP is used as a reference RNA.
Table 1.
Sequencing of the Pst1 site.