Figure 5.

The sequestosome p62 forms mitotic inclusion bodies (MIBS) enriched in CDK1-phosphorylated p62 and K63 polyubiquitin chains. (A) Representative single-plane confocal images of HeLa cells synchronized in mitosis by a double thymidine block, showing staining of p62, K63 polyubiquitin (K63-Ub), and DNA (Hoechst) in cells at different mitotic stages compared to a cell in interphase; M: metaphase; A: anaphase; I: interphase. Bar: 10 μm. (B) Deconvolved single-plane confocal images of a representative HeLa cell at metaphase from a non-synchronized cell population, showing staining of pT269-S272-p62 (CDK1-induced), pS403-p62 (stress-induced), and total p62. Enlarged views of the boxed regions emphasize MIBS spherical shape and enrichment in p62 phosphorylated at mitotic sites (T269-S272) but not at the canonical stress-induced site (S403); Bars: 10 μm or 2.5 µm. (C) Quantification of polyubiquitin clusters in non-synchronized HeLa cells at prometaphase-metaphase. Cells were transfected with control siRNA or p62-specific siRNAs and stained using anti-α-tubulin and anti-ubiquitin antibodies, means ± SE from 102 to 136 cells from at least 4 independent experiments. Statistical significance was analyzed using the Kruskal–Wallis test and Dunn′s multiple comparisons: ****, p < 0.0001. See also Figure S5A for the representative phenotype in p62-depleted cells and Figure S5B for MIBS staining in other cell types. (D) p62 or control IPs were prepared using anti-p62 or rabbit IgG, respectively, from mitotic HeLa cells (nocodazole-treated and recovered by a mitotic shake-off); cells were treated with a reversible crosslinker (DTME) or the vehicle prior to cell lysis and p62 IPs were analyzed by Western blot using anti-p62, anti-BAG3, and anti-K63 polyubiquitin.