A549 or A549s expressing H2BK-GFP cells (5 × 103) were plated into 12-well tissue culture plates and incubated for 24 or 48 hours. Wild-type H1975 EGFR cells (8 × 103) were plated into a sterile 12-well tissue culture plate and incubated for 24, 48, or 72 hours. Wild-type 16HBE or 16HBE stable cell lines (8 × 104) expressing CAR-GFP or transfected with CAR-targeted siRNA were plated into a sterile 12-well tissue culture plate and incubated for 24 or 48 hours. Hoechst was added to the medium of the cells for 30 min to stain DNA and then cells were fixed for 15 min in 4% PFA/PBS in the dark. Using a 10× objective on an Olympus IX71 inverted fluorescence microscope, cells were imaged using the same exposure with the 406-nm laser and six images per well were saved as TIFF files. H2BK-GFP A549s were grown for 24 hours, and using an Olympus IX71 inverted fluorescence microscope (10× objective), cells were imaged live using the same exposure with the 488-nm laser, six images per well were saved as TIFF files, and then the cells were incubated for a further 24 hours, after which they were imaged again. TIFF files were opened in ImageJ, and the number of nuclei per image was analyzed by thresholding the nuclei followed by selecting on the basis of size and circularity to determine the number of particles. The number of particles was counted per field, enabling the total number of nuclei to be calculated across multiple fields.

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