Johnson CE , Freel CD , Kornbluth S .

CA102707 NCI NIH HHS , R01 GM08033 NIGMS NIH HHS , R01 GM080333-01A1 NIGMS NIH HHS , R01 GM080333 NIGMS NIH HHS , R01 CA102707 NCI NIH HHS

	Figure 2. Oocyte fluorescence is due to activation of caspases(A) Oocytes were injected with the IRDye and either yCC (267 nM), CC (267 nM), CC (267 nM) + z-VAD-fmk (40 nM), or tBid (167 nM) and imaged after 1 hour. Average oocyte fluorescence is displayed in Supplemental Figure 1c. (B) Left, IRDye and 20 ng of either B-globin or flag-tagged Xenopus Bok (xBok) mRNA was injected into oocytes, allowed to express for 5 hours at room temperature and them imaged. Average oocyte fluorescence is displayed in Supplemental Figure 1d. Right, oocyte lysates immunoblotted with anti-flag antibody demonstrated xBok expression. (C) Cell-free lysates were prepared from untreated or CC-injected oocytes, and analyzed for caspase activity over time after the addition of the colorimetric caspase substrate Ac-DEVD-pNA. (D) Top, oocytes were injected with the IRDye and increasing doses of CC or yCC and imaged after 1 hour. Bottom, lysates prepared from these oocytes immunoblotted for active caspase 3.
	Figure 3. Caspases are rapidly activated in response to cytochrome c; caspases remain active for hours although their activity is only required for 10 minutes to ensure apoptosis(A) Oocytes were injected with yCC (healthy) or CC (apoptotic) at 106 nM, and photographed 3 hours later. (B) Oocytes microinjected with IRDye and either yCC or CC at 67 nM were imaged for fluorescence after 5 minutes, demonstrating rapidity of the signal in response to even small amounts of CC. Average oocyte fluorescence is displayed in Supplemental Figure 1e. (C) Oocytes microinjected with IRDye and either yCC or CC at 533 nM were imaged after 24 hours, demonstrating a stable, selective induction of signal in CC-injected oocytes even at high yCC concentrations. Average oocyte fluorescence is displayed in Supplemental Figure 1f. (D) Oocytes microinjected with either CC or yCC at 80 nM at t=0 were then injected with IRDye at various times indicated, and imaged 30 minutes later. Average oocyte fluorescence is displayed in Supplemental Figure 1g. (E) CC or yCC was microinjected at 533 nM at t=0, then IRDye and z-VAD-fmk (70 nM) were injected at times indicated and then imaged 30 minutes later. Note z-VAD-fmk-mediated signal inhibition even at high concentrations of CC. Average oocyte fluorescence is displayed in Supplemental Figure 1h. (F) Survival curve based on observation of apoptotic morphology of oocytes that had been treated as described in E.
	Figure 4. A cytochrome c threshold exists in oocytes and is lowered by Smac addition(A) Oocytes were microinjected with IRDye and either sub-threshold CC (44 nM), supra-threshold level of CC (107 nM), 44nM CC plus Smac protein, 44nM yCC plus Smac, or 44nM CC plus Smac that had been heat-inactivated by heating at 95°C for 5 minutes (HI-Smac) and then imaged after 40 minutes. Smac was injected to a concentration of 280 nM. Average oocyte fluorescence is displayed in Supplemental Figure 1i. (B) Low doses of CC were injected with the IRDye and Smac and imaged after 30 minutes. Average oocyte fluorescence is displayed in Supplemental Figure 1j. (C) Oocytes were injected with IRDye and increasing doses of CC and imaged after 45 minutes. Naive oocytes from the same batch were then injected with IRDye and 90nM CC, observed for apoptotic morphology and then 3 apoptotic and 3 living oocytes were imaged for fluorescence. Average oocyte fluorescence is displayed in Supplemental Figure 1k. (D) Lysates were prepared from the apoptotic and living oocyte groups described in C and then immunoblotted for XLX.
	Figure 5. Progesterone-induced oocyte maturation decreases sensitivity to cytochrome c(A) Reflected light images of healthy stage VI oocytes (top left), progesterone-treated oocytes demonstrating GVBD (top right), cytochrome c-injected apoptotic oocytes (bottom left), and deteriorating un-fertilized oocytes 20 hours after GVBD (bottom left). (B) Oocytes were treated with progesterone and allowed to mature until GVBD, approximately 5 hours. Post-GVBD or control-treated oocytes were then injected with the IRDye and yCC or CC (at 67 nM), and then imaged after 1 hour. Average oocyte fluorescence is displayed in Supplemental Figure 1l. (C) Oocytes were treated with progesterone for 30 minutes before injecting them or control-treated oocytes with the IRDye and yCC or CC (at 533 nM), and imaged after 1 hour. Average oocyte fluorescence is displayed in Supplemental Figure 1m. (D) Oocytes were treated with progesterone or control, microinjected with IRDye after GVBD and then imaged for fluorescence 24 hours after progesterone treatment. Oocytes injected at the same time with IRDye and CC at 67 nM were included as a positive control. Average oocyte fluorescence is displayed in Supplemental Figure 1n.
	Figure 6. Cytochrome c injection of a single blastomere causes death of the entire embryo(A) Fertilized eggs were injected prior to the first cell cleavage with the IRDye or IRDye and CC and then imaged during the 2-cell stage. Autofluorescence is shown in the 700 nm channel and caspase activity in the 800 nm channel. (B) One blastomere of a two-cell embryo was microinjected with IRDye and either CC or yCC (at 270 nM)., and imaged after 30 minutes (approximately 3 hours after fertilization). (C) Embryos from B were photographed 4 hours after fertilization. (D) Embryos were injected as indicated and then imaged 30 minutes later (approximately 3 hours after fertilization). (E) Photograph of Mos-arrested embryos, with left half arrested while right half of embryos continue.
	Figure 7. Apoptosis is not cell-autonomous in the early embryo(A) Fluorescent dextrans were injected as indicated and then embryos were imaged 30 minutes later. (B) One embryo injected as indicated and imaged 30 minutes later. Signal in the 700 nm channel represent fluorescent dextran and signal in the 800 nm channel represents IRDye cleavage. (C) Emi2 mutant protein was injected into one blastomere at the two-cell stage and photographs were taken approximately 4 hours post-fertilization. The embryo on the left demonstrates full arrest, rather than arrest of only the injected side. (D) Left, Embryos were injected as indicated (approximately 2 hours post-fertilization) and imaged for fluorescence at the indicated times after fertilization. Right, photographs of the embryos 19 hours post-fertilization, confirming IRDye-injected embryos are dividing normally and show no signs of apoptosis.

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