Deletion of cathepsins B, L, or S significantly increases tumor cell death. TUNEL staining was used to visualize apoptotic cells in tumors from control RT2 (A), CtsB−/− RT2 (B), CtsS−/− RT2 (C), CtsL−/− RT2 (D), and CtsC−/− RT2 (E) mice, stained under identical conditions. Positive cells are stained in brown, and hematoxylin (blue) is used as a counterstain. Representative images from each genotype are shown. (F) The percentage of apoptotic (TUNEL+) cells was calculated from several fields of each tumor in each mouse analyzed, and the means and standard errors are shown for each genotype. The total numbers of fields scored are as follows: RT2 controls: 209 fields from 21 mice; CtsB−/− RT2: 94 fields from 10 mice; CtsS−/− RT2: 122 fields from 12 mice; CtsL−/− RT2: 36 fields from four mice; CtsC−/− RT2: 86 fields from eight mice. The “controls” column corresponds to both +/+ and +/− RT2 littermates generated from the four cathepsin mutant/RT2 crosses. The means and standard errors are shown, and P values were calculated by comparison to the RT2 littermate control group using the Wilcoxon t-test; (***) P value of <0.0001. Bars, 50 μm.