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 Nek5 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.
