Aggregated proteins in SETX-depleted cells are localized primarily in nucleoli. (A) Cytosolic (CP), nucleoplasmic (NP), and nucleolar (No) fractions were isolated from U2OS cells with control or SETX shRNA by sucrose gradient, and each fraction was used to isolate detergent-resistant aggregates. Lysates and aggregates from different fractions were analyzed by Western blotting for PSMD2, PSMD8, β-actin (cytoplasmic markers), and fibrillarin (a nucleolar marker). (B) Three replicates of A were performed and quantified; levels of PSMD2 and PSMD8 in the aggregates of nucleolar fraction normalized by lysate levels are shown relative to control cells. (C) Localization of endogenous PSMD8 was performed by immunostaining with PSMD8 and fibrillarin antibodies in U2OS cells, with shRNA-mediated SETX depletion or mock treatment. Arrows indicate PSMD8 in nucleoli. (D) Quantification of control and SETX-depleted U2OS cells showing the percentage of cells with PSMD8-positive nucleoli in C. A total number of 165 cells were counted for the control group, and 90 cells were counted for SETX-depleted group. Error bars indicate standard error. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively, by two-tailed t test assuming unequal variance (B) and chi-square test (D), between control and SETX-deficient groups; ns = not significant. (E) Localization of endogenous SETX in U2OS cells was detected by immunostaining with SETX and fibrillarin antibodies. Source data are available for this figure: SourceData F7.