(A) Western blot analysis of protein lysates from ES cells stimulated to differentiate with RA for the indicated times. The Western blot membranes were probed with an antibody that specifically recognizes only active Caspase-3. MEF, mouse embryonic fibroblasts. (B) Alkaline phosphatase staining of ES cell colonies stimulated with RA (1 µM/ml) or coincubated with RA and the pan-Caspase blocking peptide VAD.fmk (100 µM) after 3 days. Cells incubated with leukemia inhibitory factor (LIF) served as controls. The majority of colonies lost staining due to differentiation, whereas coincubation with VAD prevented differentiation. Magnification, 10X. (C) Casp3 heterozygous (+/−) and knockout (−/−) ES cells were stimulated with RA (1 µM) for 3 days and stained for alkaline phosphatase. Casp3^+/− ES cells differentiated, whereas the majority of Casp3^−/− ES cells did not (10X). Controls were cells incubated without LIF (D, left) Quantification of colonies shown in panel B as described in Experimental Procedures. Mean values are shown. (D, right) Quantification of colonies shown in panel C. Mean values are shown. (E) Casp3^+/− and Casp3^−/− ES cells were injected into immunocompromised nude mice, and the resultant tumors were analyzed histologically. Typical aspects of Casp3^+/− and of Casp3^−/− tumors included derivatives from all three germ layers within a teratoma or immature and undifferentiated cells, respectively.

(A, top) In vitro-translated hNanog, but not its D69E mutant, is cleaved by Caspase-3. (A, bottom) Proposed caspase cleavage site at position 69, separating the N-terminal domain of Nanog from the rest of the protein. (B, top) Western blot showing expression of hNanog and mutant hNanog with or without a mutation at position 69 (D69E) that obviates cleavage. The results represent ES cells transfected with hNanog or D69E hNanog in cultures treated with RA (1 µM) or untreated. After induction of differentiation, cleaved hNanog appeared as a 27 kDa band that was not visible when the cells were transfected with the mutated form of hNanog. (B, bottom) Western blot showing expression of hNanog and mNanog and mutant hNanog and mNanog carrying a mutation at position 69 and 67 respectively (D69E, D67G) that obviates cleavage. The results represent ES cells transfected with depicted Nanog versions and treated with RA. Cleaved hNanog and mNanog appeared as a 27 kDa band that was barely visible when the cells were transfected with the mutated forms of hNanog and mNanog. (C) Western blot analysis for endogenous mNanog reveals the cleaved form of the protein after induction of differentiation with RA. (D) In vitro caspase cleavage assay reveals that Caspase-9 can effectively cleave in vitro-translated mNanog (E) Proliferation of ES cells harboring hNanog or D69E hNanog and grown under normal conditions with LIF or RA stimulation. Mean (+/− SD) levels of ³H incorporation in triplicate experiments indicate a consistently greater proliferative advantage for cells expressing the hNanog cleavage mutant. (F) Proliferation of ES cells expressing D69E or wild-type hNanog that were mixed 20:80 with EGFP-labeled ES cells. The percentage of unlabeled ES cells in the mixture was determined during growth in the absence of LIF. Cells carrying the caspase cleavage-resistant mutant (D69E hNanog) had a marked growth advantage over control or hNanog-expressing cells. The data are means (+/− SD) of triplicate experiments. (G) Mouse ES cells were plated at low densities (5000 cells/cm²) and transfected with wild-type mNanog, D67G mNanog (caspase cleavage-resistant Nanog) or control plasmid (GFP) and cultured with or without LIF for 3 days. Tranfection with D67G mNanog substantially increased the number of undifferentiated (AP-positive) colonies.

(A) The R1 ES cell line was stimulated with RA (1 µM) for the indicated times and Caspase activity was measured in an in vitro Caspase activity assay. The data are means (+/− SD) of triplicate experiments. (B) The same ES cell line was again exposed to RA for various times and the mean (+/− SD) percentage of cells undergoing programmed cell death was determined by counting apoptotic bodies . UV, ultraviolet light. (C) ES cells expressing the Caspase sensor (Casp^sensor) were stimulated with RA (1 µM), fixed at the four indicated time points and stained with an antibody against a reporter protein (enhanced yellow fluorecent protein, EYFP). Mainly cytoplasmic staining indicates low Caspase acitivty, while mainly nuclear staining typically indicates increased Caspase activity. Immunofluorescence images (40X) were taken from representative fields. Although a shift of the signal from the cytoplasm to the nucleus is apparent at 12 hours, the cells did not show any signs of apoptosis. Cells treated with stausporine at 6 hours served as positive controls; scale bar = 10 µm. (D) Similar experimental setting as in panel C except that the mean percentage of cells with mainly cytoplasmic or nuclear staining was determined. (E) Western blot analysis of nuclear lysates isolated from mouse ES cells after stimulation with RA for the indicated times. The blotted membrane was probed with an antibody against PARP-1. The uncleaved form of PARP-1 is apparent at the earlier poststimulation times, with the cleaved form (85-kDa fragment) appearing after 24 hours, indicating PARP-1 cleavage. (F) Immunostaining of a mouse ES cell colony stimulated with RA or incubated with leukemia inhibitory factor (LIF) for 2 days. The antibody used specifically recognizes the 85 kDa form of PARP-1. Magnification, 40X.

ES cells carrying a constitutively active form of Casp3 gene (Casp3rev) or a mutated version (mCasp3rev) under the control of a tetracycline-inducible element were stimulated with doxycycline at increasing concentrations. (A) Western blotting and detection of Oct4 and Nanog were then performed. Both Oct4 and Nanog disappeared when the expression of Casp3rev but not mCasp3rev increased. WT, wild-type. (B) ES cells show clear morphologic signs of differentiation when Casp3rev but not mCasp3rev expression is increased. (C) RT-PCR of Oct4, Nanog and differentiation markers. Both Oct4 and Nanog were downregulated when expression of Casp3rev but not mCasp3rev was increased, whereas expression of the differentiation markers Bmp2, Laminin B1 and Gata4 increased.