Fig 2: Failure to regulate rRNA processing with translation inhibition results in nucleolar fragmentation. (A–D) FRAP of U2OS cells stably expressing RPL7A (A, B) or Nol9 (C, D) untreated or treated with cycloheximide or puromycin to repress translation without inducing a stress response. Despite inhibition of translation, nucleolar dynamics remain unaltered indicating that previously identified alteration in nucleolar dynamics is not a result of translation inhibition. Red arrowheads denote photobleached nucleoli. (E) Northern blotting of rRNA after treatment with CHX and Puro demonstrates that 47S rRNA and 34S fragment do not accumulate. Instead, there is a reduction in 47S levels (ln 9 and 10) commensurate with the inhibition of ribosomal protein synthesis. (F) After indicated treatments, nucleoli of U2OS cells were analyzed by immunofluorescence by staining for NPM. Stress granule formation was monitored by eIF3B localization. As shown previously, NaAsO2 results in stress granule formation and maintenance of nucleolar structure. However, translational inhibitors that do not trigger a stress response (CHX and Puro) result in fragmentation of nucleoli.

Fig 3: Nucleolar structure is maintained upon activation of the Integrated Stress Response. (A) Immunofluorescence of untreated U2OS osteosarcoma cells (Aa) or treated with Actinomycin D (Ab), NaAsO2 (Ac), Lomustine H2O2 (Ad), or H2O2 (Ae). Nucleolar integrity was monitored by staining for nucleophosmin (NPM, green) and activation of the ISR was monitored by analysis of stress granule formation using eIF3B (red). (B) Nucleolar number is unaffected by stress response on a per cell basis. Cells were treated with indicated stresses and the number of retained nucleoli per nuclei were manually counted using NPM as a nucleolar marker. (C) Representative images of Imaris 3D reconstruction after indicated treatments. (D) Nucleolar volume is unaffected after ISR-activating stresses (NaAsO2 and Lomustine), while volume is significantly decreased after mTOR-inactivating H2O2 treatment and ActD, which inhibits transcription. Data analyzed by Student's t-test (* P < 0.05, **** P < 0.0001). (E) Sphericity of nucleoli was determined following 3D reconstruction. ISR-activating stresses results in a more spherical nucleolar structure, while mTOR-inactivating H2O2 treatments results in a statistically significant decrease in sphericity Data analyzed by Student's t-test (* P < 0.05, **** P < 0.0001).