Figure 5. UCH37 activity regulates proteasome condensates upon osmotic stress.

(A) HCT116 cells expressing Flag-GFP-UCH37(C88A) were treated with 0.2 M sucrose for 15 min, then fixed and immunostained with RPT6 and K48-specific antibodies. The line profile represents the magenta (RPT6), red (K48), and green (GFP) channel intensities along the arrow shown in cyan (merged panel). Scale bar, 5 μm. (B–D) RPT6 foci numbers in each cell were quantified and are shown as mean ± standard deviation (SD); n > 100 cells were measured for each cell type. Unpaired t-tests were performed between each cell type and P: ***, p < 0.001; ****, p < 0.0001; n.s., not significant. (E) Whole-cell lysates were collected from P, KO, and WT cells with or without 30 min 0.2 M sucrose treatment, and then analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) and immunblotting with the indicated antibodies. (F) Quantification of Ub conjugates from whole-cell lysates immunoblotted with FK2 antibody (for total Ub conjugates) or with linkage-specific anti-Ub antibodies. Representative blots are shown in (E). Anti-Ub signals relative to those in P were plotted after normalization using the signal intensities from total protein stain. Mean ± SD from two independent replicates are plotted. Unpaired t-tests were performed between each cell type and P: *, p < 0.05; **, p < 0.01.

Figure 5—figure supplement 1. UCH37 regulates branched Ub chain-containing inclusions induced by proteolytic stresses.

(A) Schematic of cell line constructions. UCH37 knockout (KO) was derived from parental HCT116 cells (P) by CRISPR. KO cells were then infected with retrovirus encoding wild-type, C88A, or EWI versions of Flag-GFP-UCH37, followed by stable cell line selection to create WT, C88A, and EWI polyclonal cell lines. (B) Whole-cell lysates from cells as described in (A) were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting; * denotes a nonspecific band. Note that endogenous UCH37 has two isoforms, and UCH37(C88A) is oligoubiquitinated. (C, D) Cells are treated with 0.2 M sucrose for 15 min (C) or 0.5 mM sodium arsenite for 90 min (D), fixed and immunostained with the indicated antibodies. The line profiles represent the magenta, red, and green channel intensities along the arrow shown in cyan (merged panel). Scale bar, 5 μm.