Fig. 10

SINGs correlate with embryonic lethality and impaired cell division. a Stress induces SING formation in embryos. Embryos were obtained from worms expressing GFP::Ub and mCh::H2B and placed in 500 mM NaCl for 1 h or 10 mM H₂0₂ for 30 min. Embryos formed SINGs under both types of stress conditions. Scale bar indicates 10 μm. b Hatching rate of embryos that contain SINGs. Embryos were dissected from gravid adults that were soaked in M9 (unstressed) or 500 mM NaCl (stressed) for 1 h. Stressed embryos were screened under the microscope for the presence of SINGs. Embryos with SINGs showed a 0% hatch rate versus a 100% hatch rate for unstressed embryos or stressed embryos without SINGs. A total of 40 embryos were analyzed for each condition from 3 independent experiments. Statistical significance was calculated by a Fisher’s Exact test: ****p < 0.0001. c Hatching rates of wild type and ubc-18 mutant embryos in unstressed and stressed conditions. Embryos were subjected to two different stresses: salt stress (500 mM NaCl for 60 min) and oxidative stress (10 mM H₂O₂ for 30 min). Both types of stress reduce the hatching rate of wild type embryos. However, in ubc-18 or ubc-20/ubc-22 (RNAi) embryos which have reduced SING formation, salt stress does not reduce the hatching rate. Oxidative stress does reduce hatching rate in ubc-18 and ubc-20/ubc-22 (RNAi) embryos. In ubc-18 and ubc-20/ubc-22 (RNAi) embryos, the numbers for the M9 and the 500 mM NaCl hatching rates showed no statistical difference. Experiments were repeated three times with 25 embryos per trial for a total of 75 embryos per condition. Statistical significance was calculated by a Fisher’s Exact test: ****p < 0.0001. d Time-lapse imaging on unstressed and stressed embryos expressing GFP::Ub and mCh::H2B. Ten embryos from the unstressed and ten embryos from the salt stressed groups were observed on the confocal microscope with time-lapse imaging for 15 min each. Many cells in each unstressed embryo were able to complete cell division within 5 min. Cells in salt stressed embryos showed no progression through the cell division cycle. The asterisk in the M9 embryo shows a cell that proceeds through prophase, metaphase, and anaphase of the cell cycle. The asterisk in the 500 mM NaCl embryo shows a nucleus that has SINGs and remains in prophase. The arrow shows a nucleus with SINGs that remains in metaphase throughout the 15 min observation period. A total of 10 embryos were collected from 3 independent experiments. Scale bar indicates 10 μm