CREST protein is aggregation-prone
. (A) CREST aggregates upon its accumulation in the nucleus. GFP-tagged, Flag-tagged or untagged CREST are largely confined to the nucleus where they are diffusely distributed or can form dot-like aggregates. In high-expressing cells CREST aggregates are visibly larger and the protein undergoes a shift to the cytoplasm where it also aggregates (‘large + cyt’ panel). SH-SY5Y cells expressing tagged or untagged CREST were analysed 24 hours post-transfection. (B) CREST-GFP is almost entirely confined to the nucleus where it is excluded from the nucleolus visualised by ethidium bromide (EtBr) staining. It is not enriched in PML bodies (anti-PML staining), Gems (anti-SMN staining) or Cajal bodies (anti-coilin p80 staining), but is concentrated around speckles (FISH with MALAT1 probe) and highly enriched in paraspeckles (FISH with NEAT1 probe) in low-expressing cells. (C) Nuclear aggregates formed by CREST-GFP overexpressed in SH-SY5Y cells do not overlap with PML bodies, Gems, Cajal bodies or paraspeckles but surround speckles. Arrowheads denote nuclear bodies in CREST-expressing cells. Scale bars, 10 μm.