Visualization and Quantitative Analysis of Genotoxin-Induced PARP1/PARP2 Activation in Cells Using a Fluorescent Fusion Protein-Based Reporter

Our lab studies PARP1 and PARP2-dependent DNA repair and DNA damage response pathways in normal and transformed cells. A challenge in the field is quantitative analysis of PARP1 and PARP2 activation in cells while avoiding cell lysis. This allows for subcellular localization studies.

To begin, add 375 microliters of fetal bovine serum and Penicillin-Streptomycin free DMEM to a 1.5-milliliter sterile microcentrifugation tube. Then, add 10 microliters of a lipid-based transfection reagent and all the required plasmids to the tube. Tap the microcentrifugation tube gently to mix the medium, transfection reagent, and plasmid DNA.

Incubate at room temperature for 15 to 30 minutes to allow DNA and transfection reagent complex formation. Next, add the plasmid and transfection reagent mix dropwise to the 60-millimeter dish containing the cultured 293FT cells without removing the medium and return the cells to the incubator for 48 hours. Then, collect the culture medium from the transfected 293FT cells.

To isolate the lentivirus particles, filter the collected medium through a sterile 0.45-micrometer filter to separate cell debris from the lentiviral particles. For transduction, obtain the desired cells cultured for 24 hours and verify that the cell confluency is between 20%and 40%Next, add one milliliter of virus, one milliliter of growth medium, and two microliters of Polybrene to a 15-milliliter conical tube, mixed gently by inversion. Now, remove the growth medium from the six-well plate.

Add the virus, medium, and Polybrene mix to each well. Place the cells with the transduction mix in an incubator containing a humidified atmosphere at 32 degrees Celsius and 5%carbon dioxide for 16 to 18 hours. To validate the expression of the RNF146 amino acids, 100 to 182 EGFP, seed 200, 000 LivePAR-expressing cells per culture dish containing cover slips.

Allow the cells 24 to 36 hours to adhere to the cover slips, condition the medium, and begin replicating. Then expose the LivePAR-expressing cells to the given reagents. Add the compounds directly to the medium containing the cells on the cover slips and gently swirl the medium in the cell culture dishes to distribute the compounds.

Incubate the 60-millimeter dishes at 37 degrees Celsius for 60 to 90 minutes. To fix the cells, remove the medium and wash the cells with PBS. Prefix the cells in 4%formaldehyde in PBS for 15 minutes at room temperature.

After removing the formaldehyde, wash the cells three times with PBS. Then, add three milliliters of cold methanol and acetone to the cells in a 7:3 ratio to fix them. Place the dishes at minus 20 degrees Celsius for nine minutes.

Then, remove the methanol acetone solution. After washing the cells three times with PBS, pipette 15 microliters of antifade medium containing DAPI onto a glass slide. Gently mount the cover slip onto the mounting medium with the cells facing inward, while minimizing bubbles.

Center and secure the cover slip onto the glass slide and seal the sides by applying a small amount of clear topcoat nail enamel to the edges. Place the slides in the dark to allow the nail enamel to dry. After downloading and installing Image J, open Image J and install the macro text file LivePAR_Macro by clicking on Plugins, selecting Macros, and choosing Install.

Open all confocal files in Image J and save the files as TIFF files to preserve the original files. Then, open a spreadsheet document and save it as Date_CellLineFociAnalysis. Analyze one TIFF file at a time.

Press 1 to find the nuclei. When the ROI manager window opens, select all entries and press add to overlay the nuclei area over the original image. Delete incorrectly identified nuclei and exclude nuclei that are not fully within the frame.

Then, draw the nucleus using the Freehand Selection tool and press 2 to quantify PAR foci. Select each nucleus in the ROI manager and count the number of foci per nucleus. Copy and paste the quantified data into the open spreadsheet document after scoring all nuclei in the file.

Similarly, repeat the analysis for all TIFF files. When finished, plot PAR foci per nucleus in the software of choice. Vehicle control cells showed pancellular EGFP staining.

Genotoxin treated cells showed accumulation of nuclear foci representing localization within the nucleus. Pretreatment with the PARP1 and PARP2 inhibitor veliparib resulted in the loss of PAR foci. Quantification of the number of PAR foci per nucleus as a function of time and treatment conditions showed similar results.

This protocol allows us to quantify activation of the PARP1 and PARP2 signaling axis in response to genotoxins and following genetic alterations. We have employed traditional immunofluorescence techniques with this assay to validate colocalization of proteins with poly ADP-ribose. Moving forward, we are using this assay for genotoxicity testing for high throughput analysis of PARP1, PARP2, or PARP inhibitors, and for identifying novel factors involved in PARP1 and PARP2 signaling.