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. 2020 Oct 20;295(51):17672–17683. doi: 10.1074/jbc.RA120.015398

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© 2020 Klickstein et al.

Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license.

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Figure 4. — Analysis of bortezomib-induced aggregates by AggreCount. A, HeLa Flp-in TRex cells were treated with 1 μm bortezomib (Btz) for 8 or 18 hours (hr), fixed, and stained with an anti-ubiquitin antibody to visualize ubiquitin-positive aggregates and aggresomes. The nuclei were stained with Hoechst. B, AggreCount was used to determine the average (Avg.) number of aggregates per cell (upper panel) and average aggregate area (lower panel). C, the number of aggregates in different cellular compartments was quantified. D, the area of aggregates in different cellular compartments was determined. E, prolonged bortezomib treatment (18 hr) leads to an increase in perinuclear aggresome area. At least 41 cells were analyzed. The graphs show the means ± S.E. *, p ≤ 0.1; **, p ≤ 0.05; ***, p ≤ 0.001 as determined by one-way ANOVA with Bonferroni correction (B–D) or Mann–Whitney (E). Scale bar, 10 μm.