Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a cellular system that maintains genome stability and integrity. Dysfunctions in this process cause several disease, such as cancer, aging-related disease, and chronic inflammation. Our goal is to identify proteins that cooperate in this process, which allows the identification of alternative targets for therapeutic integration.

To characterize the protein's participation in the DNA damage response, immunofluorescence studies with the phosphorylated form of the H2AX histone marker of DNA damage are usually applied. Moreover, protein collection assays such as co-immunoprecipitation can be performed after DNA damage to identify the protein's role in signaling. The interaction studies are usually based on immunoprecipitation assays, but these are in vitro and do not provide information about the location of the interaction.

Immunofluorescence can give information about the cell localization of the proteins, but proving the interaction can be difficult and depends on the resolution of the microscope. We show here that a simple protocol combining proximity ligand assay with Gamma-H2AX staining allows the spatial and temporal characterizations of proteins interaction in DNA damage context. Moreover, we have provided a user-friendly macro for data analysis.

To begin, take cultured U2OS cells with 60%to 70%confluency and split them using 0.25%Trypsin-EDTA. Prepare 60-millimeter plates by adding round 13-millimeter cover slips. Plate 400, 000 cells in a 60-millimeter plate containing multiple 13-millimeter round cover slips.

Then remove the culture medium from the plates. Add culture medium containing 25 micromolar etoposide and incubate cells for 20 minutes, one hour, and three hours. After incubation, remove the medium from the plates.

Wash the cells with PBS three times. To fix the cells, add ice-cold methanol in enough volume to cover the cover slip surface and incubate for 10 minutes at minus 20 degrees Celsius. Then wash the cover slips with PBS three times and transfer them to a humidity chamber.

After tapping off PBS, add 30 to 40 microliters of permeabilization buffer and incubate for 20 minutes at room temperature. After that, wash the cover slips three times with PBS. Then tap off the PBS from the samples.

Add 30 to 40 microliters of blocking solution provided in the PLA kit to each cover slip. Incubate the plate in a preheated humidity chamber at 37 degrees Celsius for one hour. Now dilute the primary antibodies in the antibody diluent provided in the kit.

After tapping off the blocking solution, add the primary antibody solution to each cover slip and incubate at four degrees Celsius overnight in a humidity chamber. Dilute the minus and plus probes one to five in the antibody diluent. Use combinations like donkey anti-mouse minus with donkey anti-goat plus and donkey anti-mouse minus with donkey anti-rabbit plus.

Now tap off the primary antibody solution and wash once with TBST. After tapping off the excess TBST, add 20 microliters of the PLA probe solution to the cover slips, then incubate in the preheated humidity chamber at 37 degrees Celsius for one hour. Dilute the five times ligation buffer in high purity water.

Tap off the PLA probe solution and wash once with TBST. Now add ligase to the ligation solution at 1 to 40 dilution immediately before applying it to the samples. Incubate in the preheated humidity chamber at 37 degrees Celsius for 30 minutes.

Next, dilute the five times amplification buffer in high purity water. Once the ligation solution is removed from the samples, wash the samples once with TBST. Add the polymerase to the amplification solution at 1 to 80 dilutions immediately before applying it to the samples.

Incubate in the preheated humidity chamber at 37 degrees Celsius for 100 minutes. Tap off the amplification buffer, then wash two times with SSC buffer for 10 minutes each. After washing the samples two times with PBS, add the primary antibody solution to each cover slip and incubate at room temperature for one hour.

At the end of the incubation, tap off the primary antibody solution and wash three times with TBST. Then add the secondary antibody solution to each cover slip and incubate at room temperature for 20 minutes. Afterward, wash five times with TBST, two times with SSC buffer, and two times with 0.01x SSC buffer.

Finally, acquire images from different wells or cover slips regions to avoid artifacts due to nonhomogeneous incubation and save all the channels with the same name pattern. After performing the PLA dot acquisition to localize protein interaction, open images from different conditions to adjust the parameters for analysis in ImageJ. Determine and specify these parameters along with the data in the macro program.

To remove the background, click process and subtract background. Use the preview function to test different rolling ball radii. For noise removal, click process, noise, and despeckle.

Then click process and smooth in the nucleus menu to apply the smooth function and improve filling. Now click image, then type, and select 8-bit to convert to an 8-bit image. After that, adjust the threshold by going to image, adjust, and finally threshold.

Adjust the threshold to visualize all the nuclei or dots in the image while avoiding oversaturation and structures collapsing. Also, note the values and test in different images. To convert to a mask, click process, binary, and convert to mask.

For the nucleus, click process, binary, and fill holes, followed by process, binary, and watershed. For the PLA dots, click process, binary, and watershed. For counting nuclei, measure the area or length of the different nuclei to estimate the average size.

Use an approximate value to analyze particles by clicking analyze and analyze particles. Set the size to 15-infinity and check the options show mask, display results, clear results, add to manager, and exclude on edges. To count PLA dots, measure their area or radius to estimate their average size.

For each ROI on the ROI manager, click in analyze and analyze particles. Set the size to 0.02 to 3, check the options show mask, display results, clear results, summarize, and exclude on edges. Save the results showing the number of dots per nucleus in each nucleus present in the image.

Nek4-Ku70 interaction increased in the nucleus following DNA damage after 20 minutes, one hour, and three hours of etoposide treatment compared to control and DMSO-treated cells. Etoposide treatment significantly increased Nek4 topoisomerase 2-beta interaction compared to the DMSO control. Gamma H2AX staining increased after 20 minutes of etoposide treatment and remained elevated for up to three hours, indicating sustained DNA damage response.