Fig 2: TDP-43 associates with polyribosomes in cells that are actively translating. (A and B) Representative polysome profiles are shown for MN1 cells transiently transfected with hTDP-43-V5 under either standard conditions (A) or after EDTA treatment to disrupt polysomes (B). Immunoblots for hTDP-43-V5 and ribosomal protein L7a in the indicated pooled gradient fractions are aligned to the corresponding gradients. Note the clear hTDP-43-V5 signal in deep polysome fractions under standard conditions that shifts to lighter fractions upon EDTA treatment. (C–E) MN1 cells were transfected with either pEGFP or pEGFP-L10a and used for immunoprecipitation with anit-GFP antibodies under conditions for TRAP. Immunoblotting of input and IPs reveals that ribosomal protein L7a (C), endogenous TDP-43 (D) and co-transfected TDP-43-V5 (E) all co-immunoprecipitate specifically with GFP-L10a, but not with GFP alone. (F-H) Representative polysome profiles from MN1 cells transiently transfected with GFP (F) hTDP-43-V5 (G) or hTDP-43A315T-V5 (H) are shown. Note the similarity of the profiles. (I) Quantification of the relative fraction of ribosomes in polysomes from independent experiments reveals no statistically significant difference (n = 3, s.e.m. error bars, P-value indicated, unpaired two-tailed t-test).

Fig 3: 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 4: Depletion of endogenous eIF6 does not affect basal levels of protein synthesis or ribosome biogenesis. (a and b) Representative Western blot of si Scr– or si eIF6–transfected ECs and quantification of knockdown efficiency (n = 4). (c) Representative fluorescent micrographs of si Scr– or si eIF6–transfected ECs, or cycloheximide (CHX)-treated ECs following incorporation of OPP to label nascent proteins (red) using a Click-iT assay and costaining of cell nuclei (DAPI; blue). Scale bars = 20 μm. (d) Quantification of cell fluorescence following OPP incorporation Click-iT assay (n > 30 cells across three separate experiments). (e) Representative polysome profiles from si Scr and si eIF6 A431 cells after sucrose gradient fractionation, showing the small ribosomal subunit (40S), the large ribosomal subunit (60S), and the monoribosome (80S; n = 3). (f) Representative immunofluorescent micrographs of si Scr and si eIF6 ECs showing nucleolin (red) and cell nuclei (DAPI; blue). Scale bars = 20 μm. (g and h) Quantification of nucleolar frequency per cell (g) and nucleolar area (h; n > 30 and n > 60, respectively, across three separate experiments). (i–l) Quantification of pre-rRNA by qPCR in si Scr– and si eIF6–transfected ECs relative to GAPDH (n > 3): 5.8S rRNA (i), 18S rRNA (j), 28S rRNA (k), and 45S rRNA (l). (m–q) Quantification of ribosomal protein expression in si Scr– and si eIF6–transfected ECs, representative Western blots (m) and band intensity quantification of RPL7a (n), RPL10a (o), RPL26 (p), and RPL23 (q; n > 3). Values in b, d, g–l, and n–q are mean ± SEM, and significance was determined by two-sided t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Source data are available for this figure: SourceData F1.

Fig 5: Western-blot analysis of polysomal fractions. The representative images of A254 profiles (as in Fig. 4A) and western blots of 60S ribosomal protein L7a (RsL7a), eIG4G, eIF4A, Strep-Nsp2, eIF4E2, and 4EBP in corresponding fractions (Frs) along sucrose gradients in control and Nsp2 cells grown under normal (A) and hypoxic (B) conditions. Arrows on the A254 profiles pont to the picks representing 80S. The western blot analysis of proteins was investigated in three sucrose density gradients using lysates from separate experiments. Arrow, position of Nsp2 signal