Extended data Fig. 6 |. Controls related to the disruption of nucleolar structure following Pol II inhibition.

(a,b) The disruption of NPM phase separation following Pol II inhibition (a) coincide with time points at which the levels of IGS ncRNAs greatly increased (b; mean±s.d., n=3 biologically independent experiments). At these time points, no reductions in the levels of the snoRNA U8 or Alu RNA were observed. (c-e) Treating cells with the Pol II inhibitor flavopiridol (FP), with various drugs that disrupt nucleolar morphology through unclear mechanisms (MG132, Doxorubicin), with the LLPS/nucleolus disruptor 1,6-hexanediol, or with the global translation inhibitor (Cycloheximide) revealed that only Pol II inhibition simultaneously disrupted NPM phase separation (c) and induced IGS ncRNA levels (d,e). Shown are representative anti-NPM immunofluorescence images (c) and two different visual representations of ncRNA levels as detected by RT-qPCR (d,e); n=3 biologically independent experiments). In the scatter plot (e), each circle represents the value of one IGS site from one of the three biological replicates. Scale bars, 5 μm.