Fig. 5

Ubiquitination pathway components participating in SING formation. a SINGs in uba-1 and ubc-18 mutants. Antibodies to ubiquitin and the 19S proteasome were used to stain wild type worms, a temperature sensitive mutant of uba-1, and a loss of function mutation in ubc-18. The merged images show the ubiquitin, proteasome, and DAPI channels. uba-1 worms grown at 16 °C showed the presence of SINGs (551/600 oocytes) in response to salt stress (500 mM NaCl for 60 min). However, uba-1 worms grown at 25 °C showed a reduction in SING formation (12/600 oocytes) when exposed to salt stress. SINGs were not induced in ubc-18 mutants during salt stress (0/600 oocytes). Data were collected from 3 independent experiments (n = 30 worms). Scale bar indicates 10 μm. b Worms expressing RPT-1::mCh were subjected to either control RNAi (vector) or RNAi of the ubc-20 plus ubc-22 E2 enzymes. This combined RNAi reduced the appearance of SINGs. Scale bar indicates 10 μm. c Quantification of the percentage of oocytes forming SINGs after control RNAi and chn-1 RNAi treatment as described in B. A total of 1020 oocytes were observed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a two-tailed z test: ***p < 0.001. d Worms expressing RPT-1::mCh were subjected to either control RNAi (vector) or RNAi of the chn-1 E3 enzyme. Knockdown of chn-1 reduced the appearance of SINGs. Scale bar indicates 10 μm. e Quantification of the percentage of oocytes forming SINGs after treatment as described in D. A total of 1020 oocytes were observed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a two-tailed z test: ***p < 0.001