Sequential pulse labeling of mtDNA with BrdU and EdU. F11 neuroblastoma cells are sequentially incubated with BrdU or EdU for 12 hr and then switched to the other thymidine analog for an additional 12 hr. EdU (EdU-OG-TSA, green fluorophore) and BrdU (αBrdU-TSA, red fluorophore) are compared with cell morphology (DIC). (A) F11 cells are pulsed first with BrdU for 12 hr and then with EdU for another 12 hr. (B) F11 cells are pulsed first with EdU for 12 hr and then with BrdU for another 12 hr. Under both conditions, there are mtDNAs that incorporate only BrdU (red vertical arrows), only EdU (green horizontal arrows), or both BrdU and EdU (yellow oblique arrows) signals. Bar = 10 μm.

BrdU incorporation into mtDNA of primary sensory neurons. Embryonic dorsal root ganglion (DRG) neurons are incubated with BrdU for 24 hr. Amplified BrdU signal (αBrdU-TSA; B and C in green, D in red) is compared with DNA stain (DAPI, blue) and cell morphology (DIC). (A) In the absence of BrdU, no false labeling is present after TSA immunofluorescence. (B,C) BrdU is clearly incorporated into mtDNA (arrows) of DRG neurons. Asterisks in B identify DRG nuclei. (D) BrdU incorporation into mtDNA (arrows) is present in the soma and neurites of DRG neurons. Bar = 10 μm.

Signal amplification of 5-bromo-2-deoxyuridine (BrdU) incorporated into mitochondrial DNA (mtDNA). F11 neuroblastoma cells are incubated with BrdU for 12 hr. BrdU signal (αBrdU, green) is compared with DNA stain [4′,6-diamidino-2-phenylindole (DAPI), blue] and cell morphology [differential interference contrast (DIC) image]. (A) Standard immunofluorescence shows nuclear BrdU (arrowhead) signal but weak or no signal in mtDNA (arrow). (B) Reduction in background fluorescence (Image-iT) refines the visualization of BrdU signal in nuclear DNA (arrowhead) and mtDNA (arrows). (C) Tyramide signal amplification (αBrdU-TSA) significantly increases the BrdU signal in nuclear DNA (arrowhead) and mtDNA (arrows). (D) Cells that are not incubated with BrdU show no false labeling in cells after TSA immunofluorescence. Bar = 10 μm.

Inhibitors of nuclear (α) or mitochondrial (γ) DNA polymerases differentially block BrdU incorporation. F11 neuroblastoma cells are incubated with BrdU for 12 hr with or without the addition of DNA polymerase inhibitors. Amplified BrdU signal [αBrdU-TSA, (A,B in green; C,D in red)] is compared with DNA stain (DAPI, blue) and cell morphology (DIC). (A,C) Cells incubated with BrdU show both nuclear DNA (arrowheads) and mtDNA (arrows) incorporation. Insets (A,B) show magnified views of the boxed areas to illustrate the detail of the BrdU signals. (B) Addition of 7 μM aphidicolin (+APH) inhibits nuclear incorporation of BrdU, whereas mtDNA labeling (arrows) is maintained. (D) Addition of 1.0 μg/ml of ethidium bromide (+EtBr) inhibits incorporation of BrdU into mtDNA but not into nuclear DNA (arrowhead). Bar = 10 μm.

Inhibitors of nuclear (α) or mitochondrial (γ) DNA polymerases differentially block EdU incorporation. F11 neuroblastoma cells are incubated with or without DNA polymerase inhibitors 4 hr prior to and together with EdU for an additional 12 hr. Amplified EdU signal (EdU-OG-TSA) is compared with DNA stain (DAPI, blue) and cell morphology (DIC). (A) Cells incubated with EdU show both nuclear (arrowheads) and mtDNA (arrows) incorporation. (B) Addition of 7 μM aphidicolin (+APH) inhibits nuclear incorporation of EdU, whereas mtDNA labeling (arrows) is maintained. (C) The presence of 1.0 μg/ml ethidium bromide (+EtBr) inhibits incorporation of EdU into mtDNA but not into nuclear DNA (arrowheads). (D) 2′,3′-Dideoxycytidine (+ddC, 200 μM) also inhibits mtDNA but not nuclear incorporation of EdU (arrowheads). Bar = 10 μm.

Amplified EdU signal in mtDNA is associated with mitochondria and other cell-specific markers. (A) SH-SY5Y neuroblastoma cells are incubated with EdU for 6 hr. Amplified EdU signal (EdU-OG-TSA, green) is compared with DNA stain (DAPI, blue) and a mitochondrial marker, pyruvate dehydrogenase (αPDH, red). EdU signal (arrows) localizes to mitochondria. Lower panels in A are deconvolved signals and demonstrate a clear association of mtDNA within mitochondria. (B,C) Embryonic or adult DRG neurons are incubated with EdU for 24 hr. EdU signal (EdU-OG-TSA, green) is compared with DNA stain (DAPI, blue) and cell morphology (in B, DIC). (B) EdU signal is present in mtDNA (arrows) of embryonic DRG neurons (EmDRG). (C,D) EdU incorporation into mtDNA (arrows) is present in adult DRG neurons (AdDRG), identified by a pan-neuronal marker (C, αTuj1, red) or a subtype-specific marker (D, αtrkA, red). (E) The number of EdU-positive mtDNAs per embryonic DRG soma is comparable to the number of BrdU-positive mtDNAs after 24-hr incubation (white bars). Adult DRG neurons have significantly more EdU-positive mtDNAs (black bar) compared with embryonic DRGs. Error bars indicate mean ± SEM for 20 DRG somas per group, *p<0.05. (F) mtDNA replication is more active in embryonic than in adult DRG neurons. Error bars indicate the mean ± SEM number of EdU-positive mtDNAs corrected for cytosolic volume (soma volume – nucleus volume). Embryonic DRG somas (white bar, n=20) have significantly more mtDNA replications per cytosolic volume compared with adult DRGs (black bar, n=20), **p<0.01. Bar = 10 μm.