Fig 2: Effects of bortezomib and b-AP15 on morphology and phenotype of iDCs. Peripheral blood adhering monocytes were cultured with IL-4 and GM-CSF for six days and subsequently incubated with DMSO, bortezomib or b-AP15 for further 24 hours. Thereafter, cells were harvested and generated iDCs were analyzed using PCR, western blot, microscopy and flow cytometry. (A, B) Expression of USP14 and UCHL5 in monocyte-derived iDCs was determined by PCR analyses of total mRNA (derived from three different donors, using 18S RNA as control; A) and by means of western blot analyses of cell lysates (derived from two different donors using specific USP14 and UCHL5 antibodies, cofilin serving as loading control; B). (C) Photomicrograph depicts iDCs treated with DMSO (left), 10 ng/mL bortezomib (middle) and 1000 nM b-AP15 (right). Scale bar 40 µm. (D) iDCs were exposed to bortezomib and the indicated concentrations of b-AP15 for 24 h. DMSO and staurosporine served as control. The percentage of live (Annexin V-/7-AAD-) iDCs was determined by FACS with Annexin V/7-AAD staining. Combined data of five independent experiments with the indicated drug treatments are shown and compared to vehicle control DMSO (Mean ± S.E.M.). Student's t test was used to determine statistical significance (*P < .05). (E) Effects of treatment on iDC size and granularity were examined and compared to the DMSO vehicle control. Exemplarily, one representative out of at least five similar experiments is depicted. (F, G) Expression of the indicated cell surface markers characterizing iDC phenotype was assessed after exposure to bortezomib or b-AP15 and contrasted to vehicle control DMSO. Levels of expression are shown as SFI. Results of single data (F) or pooled data consisting of at least five independent experiments (G) are shown. Matched isotype controls are presented as open histograms. (Student's t test, Mean ± S.E.M., *P < .05).

Fig 3: Retention of VLX1570 and target engagement after drug wash-out.(a) OPM-2 and OPM-2-BZR cells were exposed to VLX1570 for 1 hour and the uptake of 3H-VLX1570 was determined immediately or after an additional 17 hours incubation in drug-free medium; (b) Thermostabilization of USP14 was determined 17 hours after wash-out of VLX1570 by CETSA. Equal number of cells were used at each time point (loading controls are not relevant in this type of analysis23), (c) DUB inhibition was determined by Ub-VS labeling of cell extracts after 1 h exposure to VLX1570 and after 1 h exposure followed by 17 hours wash-out.

Fig 4: Effects of bortezomib and b-AP15 on morphology and phenotype of mDCs. Peripheral blood adhering monocytes cultured with IL-4 and GM-CSF for six days were subsequently incubated with DMSO, bortezomib or b-AP15 according to the indicated concentrations for further 24 hours. LPS was added for the last 16 hours of incubation in order to induce maturation. Thereafter, cells were harvested and generated mDCs were analyzed using PCR, western blot, microscopy and flow cytometry. (A, B) Expression of USP14 and UCHL5 in monocyte-derived mDCs was determined PCR analyses of total mRNA (derived from three different donors, using 18S RNA as control; A) and by means of western blot analyses of cell lysates (derived from two different donors using specific USP14 and UCHL5 antibodies, cofilin serving as loading control; B). (C) Photomicrograph depicts mDCs treated with DMSO (left), 10 ng/mL bortezomib (middle) and 1000 nM b-AP15 (right). Scale bar 40 µm. (D) mDCs were exposed to bortezomib and the indicated concentrations of b-AP15 for 24 h. DMSO and staurosporine (Abcam) served as control. The percentage of live (Annexin V-/7-AAD-) mDCs was determined by FACS with Annexin V/PI staining. Combined data of five independent experiments with the indicated drug treatments are shown and compared to vehicle control DMSO (Mean ± S.E.M.). Student's t test was used to determine statistical significance (*P < .05). (E) Effects of treatment on mDC size and granularity were examined and compared to the DMSO vehicle control. Exemplarily, one representative out of at least five similar experiments is depicted. (F, G) Expression of the indicated cell surface markers characterizing mDC phenotype was assessed after exposure to bortezomib or b-AP15 and contrasted to vehicle control DMSO. Levels of expression are shown as SFI. Results of single data (F) or pooled data consisting of at least five independent experiments (G) are shown. Matched isotype controls are presented as open histograms. (Student's t test, Mann–Whitney U test, Mean ± S.E.M., **P < .01).

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 a1-7 (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 a1-7 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