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Quantification of DNase type I ends, DNase type II ends, and modified bases using fluorescently labeled ddUTP, terminal deoxynucleotidyl transferase, and formamidopyrimidine-DNA glycosylase

Reduction of apurinic/apyrimidinic (AP) sites

Fpg-negative deoxyribitol samples were generated by incubation in 25 mM NaBH4/70% methanol for 30 min.

2,4-Dinitrophenylhydrazine derivatization and labeling of AP sites

Fpg-negative dinitrophenylhydrazone samples were prepared in 15 mM 2,4-dinitro-phenylhydrazine (DNP-H)/2.5 M HCl for 30 min. After incubation with 10% horse sera (Invitrogen, Carlsbad, CA, USA) for 1h and washing, dinitrophenyl (DNP) was imaged using a rabbit anti-DNP primary antibody and donkey anti-goat FITC-labeled secondary antibody (Invitrogen).

H2A.X and CD3-ε labeling

After incubation with 10% horse sera and washing, the presence of Ser139-phosphorylated H2A.X histone was imaged using an FITC-labeled monoclonal antibody (Biolegend, San Diego, CA, USA). Immune cell marker CD3-ε (6) was imaged using an FITC-labeled monoclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Rat mammary gland

Slides containing 5 µm–thick formalin-fixed paraffin-embedded (FFPE) slices of rat mammary gland on day 1 and day 7 of involution [a model system of apoptosis (7-9)] were purchased from Zyagen (San Diego, CA, USA). Sudan Black was used to remove autofluoresence signals, as suggested by Romijn (10); 0.3% Sudan Black/70% EtOH was applied to the slide for 10 min and washed eight times in 1× PBS.

U87 cells

U87 human glioblastoma were grown in MEM with penicillin/streptomycin and 10% FBS. Following 3 weeks of growth, 5 mL cells were plated into slide chambers (Lab-Tek, Rochester, NY, USA) at 2 × 105 cells/mL and grown for 24h. The media was removed, and 2 mL fresh media [which contained 25 µM temozolomide, 2.5 µM carmustine, 250 µM irinotecan (all from AXXORA, San Diego, CA, USA), 1 mM H2O2, and 300 µM paraquat] or an ethanol vehicle control was added. These concentrations were chosen since they represent the median lethal dose (LD50) of these cells measured over a 24-h incubation period. Twenty-four hours later, cells were fixed with 2% PFA and permeabilized in 0.1% Triton X-100.

Epifluorescence microscopy

The signal was acquired using a Nikon Eclipse TE2000-E fluorescent microscope (Nikon, Melville, NY, USA) equipped with a CoolSnap ES digital camera system (Roper-Scientific, Trenton, NJ, USA) containing a charge-coupled device (CCD)-1300-Y/HS 1392 × 1040 imaging array cooled by a Peltier device. Images were recorded using Nikon NIS-Elements software (Nikon).

Microscopic calculations

The pixel dimensions of our microscope/camera have been calibrated by a representative of the manufacturer. At 100× magnification, each pixel element represented an interrogated area of 0.061 µm × 0.061 µm. At 40× magnification, the interrogated area per pixel is 0.162 µm × 0.162 µm. Given that 1000 L = 1 m3, for each 1 µm of sample depth, the pixel volume at 100× is 3.7 × 10−18 L (1 × 10−6 × 6.1 × 10−8 × 6.1 × 10−8 m3) and at 40× is 2.62 × 10−17L (1 × 10−6 × 1.62 × 10−7 × 1.62 × 10−7m3). Therefore, in a 6-µm phantom slice, there are ~13.4 molecules/µM at 100× magnification, whereas at 40× magnification, each pixel interrogates ~95 molecules/µM.

Results and discussion

Measuring 3′OH and 3′PO4 ends using ddTUNEL and CIAP-ddTUNEL assays

Figure 1 shows the mechanism by which the ddTUNEL assay eliminates polymer formation and stoichiometrically labels each 3′OH end with a ddU 3′H. DNase type II enzymes are associated with caspase-independent calpain/serpin– driven apoptosis (11-13). The 3′PO4 ends that result from DNase type II activity are not substrates for TdT. The sequential usage of two rounds of ddTUNEL, prior to and following incubation with CIAP, allows the determination of both 3′OH and 3′PO4 ends, giving the steady-state levels of both DNase type I and type II DNA damage (Supplementary Figure S1).





Detection of modified bases and AP sites using Fpg-ddTUNEL assay

DNA is under continuous attack from oxidative/nitrosative stressors, as well as from acylation agents. In living cells, modified DNA bases are repaired by a variety of enzymatic pathways. In Escherichia coli, the enzyme Fpg excises many types of modified DNA bases from double-stranded DNA. Fpg identifies a modified base and, initially, generates an AP site and then hydrolyzes the deoxyribose of each AP site to form a DNA nick flanked by 3′PO4/5′PO4 (14-20). Treatment of a sample with Fpg and CIAP produces a 3′OH at each Fpg-sensitive DNA base and AP site.

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