Fig 2: Structure determinants of the USP14-TRIM11 interaction. a Top: schematic diagram of USP14. The ubiquitin-like (UBL) and ubiquitin-specific protease (USP) domains, as well as the numbers of amino acids, are indicated. Bottom: interaction of the USP14 UBL domain with TRIM11. Beads-conjugated GST or GST-USP14 (2 μg each) was incubated with Flag-TRIM11 (1 μg). The input and beads-bound proteins were analyzed by western blot with anti-Flag antibody or Ponceau S staining. Asterisk indicates GST and GST fusions. b Schematic diagram of TRIM11 and its mutants. In TRIM11-2CA, the conserved Cys16 and Cys19 were mutated to Ala. Each construct was tagged with Flag epitope. c, d Interaction of Flag-TRIM11 proteins with endogenous USP14 in HCT116 cells was analyzed by co-IP assay. e Interaction of USP14 (2 μg) with GST, GST-TRIM11, and GST-TRIM11-2CA (1 μg each) was analyzed by in vitro pull-down assay. f Cell Lysate fractions enriched with the proteasomes were isolated from control, Flag-TRIM11-expressing, and Flag-TRIM11-2CA-expressing HCT116 cells, and analyzed by native gel electrophoresis and immunoblotting using α1-7 and USP14 antibodies. g Chymotrypsin-like proteasome activity in control, Flag-TRIM11-expressing, and Flag-TRIM11-2CA-expressing HCT116 cells. Slopes relative to that of vector are also shown (mean ± SEM, n = 8). h, i Half-life of YFP-CL1 in control, TRIM11-expressing, and TRIM11-2CA-expressing HCT116 cells. Representative western blots (h) and the relative YFP-CL1/tubulin ratios (h, i) are shown. The exposures of the corresponding control and TRIM11/TRIM11-2CA-expressing blots were adjusted to achieve comparable levels of YFP-CL1 at time 0. j Levels of YFP-CL1 in control, TRIM11-expressing, and TRIM11-2CA-expressing HCT116 cells grown under normal and heat stress conditions. In g and i, data represent the mean ± SEM (n = 3 unless otherwise indicated). **P < 0.01; ***P < 0.001. Uncropped blots are presented in Supplementary Fig. 13. n.s.: not significant

Fig 3: Opposing roles of TRIM11 and USP14 in tumorigenesis. a, b HCT116 cells stably expressing the indicated shRNAs (a) or proteins (b) were subcutaneously injected into nude mice. Shown are average tumor volumes over time (mean ± SEM, n = 4). *P < 0.05; **P < 0.01; ***P < 0.001. c, d Representative pictures of IHC (c) and statistical data (d) of TRIM11 expression in different stages of clinical colon cancer and adjacent normal tissues. Scale bar, 50 μm. P value was assessed using two-tailed Student’s t tests. e Kaplan–Meier analysis of cumulative survival probability of colon cancer subdivided by TRIM11 expression. The statistical significance (P = 0.0046) was assessed using log-rank test according to colon cancer patients with low or high expression of TRIM11. TRIM11 low expression, n = 28; TRIM11 high expression, n = 32

Fig 4: Role of the TRIM11-USP14 axis in cell growth and apoptosis. a Levels of endogenous TRIM11 in HCT116 cells grown at 43 °C for 0 or 90 min, or at 43 °C for 90 min, and then at 37 °C for 60 or 120 min. b, c Levels of endogenous TRIM11 (b), or Flag-TRIM11, the indicated proteasome subunits, and USP14 (c) in the SN and P fractions of HCT116 cells with and without heat shock at 43 °C for 90 min. d, g TRIM11 overexpression (d) or knockdown (g) HCT116 cells and the corresponding control cells grown under unstressed, heat shock, and heat shock plus recovery conditions were analyzed for K48 polyUb conjugates and caspase-3 activation. e, f, h Apoptosis in TRIM11 overexpression (e, f) or knockdown (h) HCT116 cells, and the corresponding control HCT116 cells, treated with and without heat shock. i, j Caspase-3 activation (i) and apoptosis (j) in HCT116 cells expressing the indicated proteins and grown under normal or heat stress conditions. k–m K48 polyUb conjugates, caspase-3 activation (k), amyloid-like fibrils (l), and apoptosis (m) in HCT116 cells expressing the indicated proteins and grown under normal and heat stress conditions. In e, f, h, j, l, and m, data represent mean ± SEM (n = 3 unless otherwise indicated). *P < 0.05; **P < 0.01; ***P < 0.001. Uncropped blots are presented in Supplementary Fig. 14

Fig 5: TRIM11 binds to both PSMD2 and USP14 and inhibits their interaction. a Control and Flag-TRIM11-expressing HCT116 cells were analyzed by western blot with the indicated antibodies. b Lysate fractions enriched in proteasomes (pellets of 5 h, 100,000×g centrifugation) from control and Flag-TRIM11-expressing HCT116 cells were analyzed by native gel and immunoblotted with antibody against the 20 S subunit α1-7 (left) or the 19 S subunit PSMD2 (right). c, h Control, Flag-TRIM11-expressing (c), and Flag-USP14-expressing (h) HCT116 cell lysates were incubated with beads conjugated with anti-Flag antibodies. Immunoprecipitates (IPs) were analyzed by mass spectrometry. Shown are spectral counts of peptides derived from the indicated proteins relative to those in the control IPs (c, h) and Coomassie blue staining of IPs (c, inset). d Interaction of Flag-TRIM11 and endogenous PSMD2 in HCT116 cells analyzed by co-immunoprecipitation (co-IP) assay. e, f Interaction of Flag-TRIM11 (e) and endogenous TRIM11 (f) with endogenous USP14 in HCT116 cells. g Interaction of USP14 with TRIM11 was analyzed by an in vitro pull-down assay using purified GST or GST-TRIM11 immobilized on beads (1 μg) and Flag-USP14 (2 μg). i, j Interaction of endogenous USP14 and PSMD2 in control and TRIM11-overexpressing cells was analyzed by co-IP assays using anti-USP14 (i) or anti-PSMD2 (j) antibody. k Cell Lysate fractions enriched with the proteasomes were isolated from control and TRIM11 knockdown HCT116 cells and analyzed by native gel electrophoresis and immunoblotting using α1-7 and USP14 antibodies. l Proteasomes purified from control and TRIM11-overexpressing HCT116 cells (++, 1 μg; +, 0.5 μg) were analyzed by native (left) and SDS (right) PAGE, followed by western blot with the indicated antibodies. Uncropped blots are presented in Supplementary Fig. 11