Fig. 4

SING formation requires ubiquitination and proteasome activity. a Worms expressing RPT-1::mCh were subjected to uba-1 RNAi for 24 h prior to soaking in 500 mM NaCl for 60 min. RPT-1::mCh does not localize to SINGs when uba-1 is knocked down. Scale bar indicates 10 μm. b The percentage of oocytes forming SINGs after uba-1 RNAi treatment as described in A. A total of 1020 oocytes were analyzed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a two-tailed z test: ***p < 0.001. c Unconjugated ubiquitin does not localize to SINGs. A worm strain expressing GFP::Ub (green) and mCh::H2B (red) shows localization of GFP::Ub to SINGs after soaking in 500 mM NaCl for 60 min. Whereas, GFP::UbAA does not localize to SINGs. GFP::UbAA lacks a C-terminal diglycine that is required for ubiquitin conjugation. Scale bar indicates 10 μm. d The percentage of oocytes forming SINGs in control and salt stress treated worms as described in C. GFP::Ub control and GFP::UbAA control or salt stressed worms showed no signs of SING formation, whereas, GFP::Ub salt stressed did exhibit SING formation after stress exposure. A total of 510 oocytes were analyzed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a Fisher’s Exact test: ****p < 0.0001. e Young adult C. elegans expressing GFP::Ub and RPT-1::mCh were either soaked in M9 buffer or 500 mM NaCl for 60 min. Worms soaked in M9 did not form SINGs. Under salt stress conditions, worms without the proteasome inhibitor MG132 formed SINGs, whereas, in the presence of the proteasome inhibitor MG132, SING formation was reduced. Scale bar indicates 10 μm. f Proteasome inhibitors MG132, bortezomib, and lactacystin inhibit the formation of SINGs in response to salt stress. The percentage of oocytes forming SINGs after proteasome inhibitor treatment. A minimum of 240 oocytes were collected from 3 independent experiments (n = 24 worms). Statistical significance was calculated between salt stress and salt stress with proteasome inhibitor by a Fisher’s Exact test: ****p < 0.0001