DNA damage, microtubule stress, and ectopic p53 activation induce senescence and engulfment of entire cells. (A and B) Doxorubicin (DOXO)-treated, GFP-expressing 4226 (A) or MCF-7 (B) cells were co-plated on a glass coverslip with proliferating, mCherry-expressing cells. 7 d later, coverslips were fixed with 4% formaldehyde and stained for DAPI. Confocal images were captured, and z-stack projections are shown adjacent; representative images are shown. Scale bar represents 10 µm (A) and 20 µm (B). Z-stack projections display 76 images taken 0.2 µm apart totaling 15 µm for A, and 14 images taken 1 µm apart with a total z range of 12.62 µm for B. (C) Mixed populations of GFP- and mCherry-expressing 4226 cells were plated simultaneously at a 1:8 ratio. When near confluence, cells were treated with DMSO, 0.75 µM doxorubicin (DOXO), 10 nM paclitaxel (pacl.), or both and washed off after 24 h. Nutlin to a concentration of 15 µM was applied every other day until fixation and imaging. Average of four independent experiments is shown, error bars indicate SEM; one-way ANOVA and Newman–Keuls posttest was used to determine significance; *, significant compared with DMSO treated. (D) MCF-7 cells were treated as in C using 0.25 µM doxorubicin, 5 nM paclitaxel, and 2.5 µM nutlin. Average of five independent experiments is shown; error bars indicate SEM; one-way ANOVA and Newman–Keuls posttest were used to determine significance; *, significant compared with DMSO treated, P < 0.05. (E and F) 4226 and MCF-7 cells were left untreated or treated with 0.75 and 0.25 µM doxorubicin, respectively, for 24 h. Either NT GFP-expressing cells were co-plated with NT mCherry cells (NT, gray bar) or doxorubicin-treated GFP-expressing cells were co-plated with NT, proliferating mCherry cells (DOXO, purple bars). 7 d after co-plating, cells were fixed and DAPI stained, and random fields were imaged by microscopy. Data shown are the percentage of GFP-expressing cells examined that had one or more DAPI-positive, red vesicles inside them. Average of three independent experiments shown; error bars indicate SEM; significance determined using unpaired t test; **, P < 0.001 (E); **, P < 0.005 (F). (G) 4226 cells were treated and plated as in F. 48 h before scoring, cultures were treated with DMSO or 20 µM QV-D-Oph, a pan-caspase inhibitor. Average of three experiments are shown; not significant by unpaired t test, P = 0.46. (H) Confocal z-stack image of MCF-7 as in B. Representative image of a cell with multiple engulfed cells is shown. Z-stack projections display 12 images taken 2 µm apart with a total z range of 21.5 µm. Scale bar represents 20 µm. (I) Representative image of a senescent 4226 cell plated with NT 4226 cells as in F showing multiple engulfed cells. Scale bar represents 10 µm. (J) Senescent MCF-7 cells were treated with doxorubicin and paclitaxel, as in D. Representative image of multiple sequential engulfments shown. Scale bar represents 50 µm. (K) GFP-expressing MCF-7 cells were treated with 0.75 µM doxorubicin or not on day 0 for 24 h, and then plated on day 1 with NT mCherry-expressing cells. On day 3, the 24-well plate was placed on an IncuCyte and imaged every 2 h until day 11. In cultures of NT mCherry cells mixed with either treated GFP cells (purple line) or NT GFP cells (black line), four different random fields of view were quantified once each day for mCherry cells within GFP cells. Error bar represents SEM; significance was determined by unpaired t test on each day; *, P < 0.05; nonsignificant P values are shown in the figure. (L) Mixed cultures of GFP- and mCherry-expressing MCF-7, MPE600, and 4226 cells were plated and doxorubicin-treated as in C and imaged for fluorescence 7 d later on a Cytation, followed by fixation, SA-βGAL staining, and repeat imaging in color bright field. Shown are examples of matching sequential images of the same cell in fluorescence and color bright field. Arrowheads indicate engulfed cells visible in both images. Scale bars represent 100 µm.