(A) Immunohistochemistry shows abundant labeling of the proliferation marker Ki-67 (DAB, brown staining) in both WT (+/+) and TRAIL-R–deficient (+/– and –/–) lymphomas. Immunohistochemical staining (A) for cleaved caspase-3 and quantification thereof (B) shows fewer median number of cells expressing active caspase-3 (DAB, brown staining) in TRAIL-R^+/– and TRAIL-R^–/– lymphomas compared with that of WT lymphomas (Mann-Whitney U test, P < 0.05). (C) LOH was detected by PCR analysis of DNA isolated from Eμ-myc TRAIL-R^+/– lymphomas in 20% (2 of 10) of the lymphomas (data not shown). (D) RT-PCR analysis on RNA isolated from Eμ-myc lymphomas of different genotypes shows decreased TRAIL-R mRNA expression in Eμ-myc TRAIL-R^+/– lymphomas compared with RNA isolated from WT Eμ-myc lymphomas. (E) Relative quantitative RT-PCR analysis and densitometry shows that loss of 1 TRAIL-R allele reduced the mean (± SEM) TRAIL-R expression to approximately 60% that in WT lymphomas (n = 4 each genotype; Student’s t test, P < 0.05). (F) Immunofluorescence (FITC, green) on living Eμ-myc lymphoma cells of different TRAIL-R genotypes using the MD-5 antibody suggest expression on WT Eμ-myc lymphoma cells (α–TRAIL-R) with a heterogenous (speckled) membrane distribution as evident from the 2 different focal images. However, expression was barely detectable in TRAIL-R^+/– Eμ-myc lymphoma cells and not present in TRAIL-R^–/– Eμ-myc lymphoma cells. Representative images are shown from 2 independent lymphomas per genotype. Original magnification, ×100. (G) Flow cytometry analysis on Eμ-myc lymphoma cells suggests expression of TRAIL-R in WT cells but not TRAIL-R^+/– and TRAIL-R^–/– Eμ-myc lymphoma cells. Eμ-myc lymphoma cells from at least 2 different animals/genotype were analyzed.