Effect of LY on cells with inactivated pRb pathways. (a) Response of cell lines with lesions in the pRb pathway to LY. NIH3T3-L1 cells expressing SV40 large T antigen (LT3) and NIH3T3-L1 cells expressing a point mutant of SV40 large T antigen unable to bind to pRb-like proteins (K1) were treated with 1.5 μM LY. Cell numbers were determined at the indicated time points in triplicates. (b) Morphology of NIH3T3-L1 cells after LY treatment. Phase-contrast microscopy was performed after incubation of NIH3T3-L1-K1 (K1) and NIH3T3-L1-LT3 (LT3) cells in 1.5 μM LY for 3 days (magnification, ×100). (c) Quantification of early stages of apoptosis. K1 and LT3 cells (control cells and cells treated with LY for 48 hours) were subjected to double staining with anti-annexin V and PI to discriminate living, early apoptotic, and late apoptotic/necrotic populations and were analyzed by flow cytometry. The percentage of PI-negative, annexin V–positive cells is shown. (d) Induction of apoptosis by LY. HeLa cells expressing a histone H2B-eGFP fusion protein (kindly provided by G. Wahl) were treated with 2 μM LY for 48 hours, and the chromatin morphology in living cells was analyzed at a wavelength of 495 nm. (e) Quantification of apoptosis by detection of cells with sub-G₁ DNA content. HeLa cells were treated for 60 hours with LY. DNA content was determined after PI staining as described in the Methods.