1. Volume 24, Issue 3, Pages 732-743 (July 2018)
Dual Function of USP14 Deubiquitinase in Cellular Proteasomal Activity and Autophagic Flux 
Eunkyoung Kim, Seoyoung Park, Jung Hoon Lee, Ji Young Mun, Won Hoon Choi, Yejin Yun, Jeeyoung Lee, Ji Hyeon Kim, Min-Ji Kang, Min Jae Lee 
Cell Reports 
Volume 24, Issue 3, Pages (July 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 732-743DOI: (10.1016/j.celrep.2018.06.058)
Copyright © 2018 The Authors Terms and Conditions

3. Figure 1
Inhibition of USP14 Increased the Cellular Proteasome Activity and the Level of LC3-II
(A) HEK293 cells were transfected with 10 nM siRNA for silencing USP14 (siUSP14) or UCHL5 (siUCHL5). A scrambled sequence was used as a control (siControl). Protein samples were collected 48 hr post-transfection and analyzed by SDS-PAGE and immunoblotting (IB). β-Actin served as a loading control.
(B) Endogenous LC3-II levels were significantly elevated in USP14−/− MEFs compared with WT MEFs. Short exp and long exp; short and long exposures of the blot, respectively.
(C) HEK293 cells were cotransfected with plasmids expressing GFP-LC3 and siRNA against USP14. Transiently overexpressed LC3 and endogenous USP14 levels were examined 2 days post-transfection.
(D) HEK293 cells were sequentially transfected with USP14 siRNA and GFP-LC3 at a 24-hr interval. After 24 hr, a significantly higher number of GFP-LC3 puncta was observed in siUSP14-transfected cells than in siControl-transfected cells. Representative images are shown after counterstaining with DAPI.
(E) Quantification and comparison of GFP-LC3 puncta numbers in (D). The plotted values are mean ± SD after normalization to DAPI-based cell numbers from more than three independent experiments.
(F) The proteasome activity measured in three independent primary USP14−/− MEF clones at passage #2 was higher than that in WT MEFs. Values plotted are the mean ± SD (n = 3).
(G) HEK293 cells were incubated with 0, 5, 10, 25, 50, or 75 μM IU1 and analyzed by SDS-PAGE followed by IB. LC3-II band intensities were quantified in three independent experiments (mean ± SD).
(H) LC3-II levels significantly increased in the presence of the USP14 inhibitor IU1. The cells were treated with 0, 25, 50, 75, or 100 μM IU1 for 8 hr.
(I) Same as in (H), except that the experiment was conducted using 100 μM IU1 with different incubation periods as indicated.
(J) IU1 treatment significantly elevates LC3-II levels in WT MEFs but not in USP14−/− MEFs. The cells were treated with 0, 25, 50, 100, or 200 μM IU1 for 6 hr. All samples were run on the same gel but IB for LC3 using 25 μM IU1 samples were removed (black line).
(K) Same as in (F), except that this suc-LLVY-AMC hydrolysis assay was performed on immortalized WT and USP14−/− MEFs after treatment with 100 μM IU1 for 6 hr. Hydrolysis reactions were run for the indicated periods. All values are represented as mean ± SD from three independent experiments.
(L) Lysosomal pH was monitored by staining with LysoTracker and pHrodo (75 nM for 2 hr) with or without IU1 treatment (100 μM for 8 hr). The average number of LysoTracker- or pHrodo-positive puncta per cell is presented as mean ± SD from three independent experiments (n = 3; in each experiment, ∼100 cells were analyzed).
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4. Figure 2
Inhibition of USP14 Impedes Autophagy Flux at the Autophagosome-Lysosome Fusion Step
(A) UPS14 inhibition (50 μM IU1) increased the LC3-II accumulation by autophagic induction. Protein samples were collected 4 hr IU1 post-treatment after additional 30-, 60-, or 120-min incubation in an amino-acid-depleted (-AA) medium. SDS-PAGE followed by IB was performed with the indicated antibodies. β-Actin served as a loading control. Veh, vehicle (0.1% DMSO). Samples from 150-min -AA incubation were run on the same gel but removed (black lines).
(B) IB analysis of transiently overexpressed LC3 in WT and USP14−/− MEFs. Cells were treated with the proteasome inhibitor cocktail (PI; 0.1 μM PS341 and 1 μM MG132) for 12 hr.
(C) The increase of LC3-II levels by the treatment with 100 nM bafilomycin A1 (BafA1) was attenuated when HEK293 cells were transfected with 10 nM siRNA for silencing USP14 (siUSP14), but not when a scrambled sequence was used as a control (siCtrl). Different sets of siRNA (both siCtrl and siUSP14) were used and the lanes running the samples were removed (black line).
(D) Contrary to the observation in WT MEFs, endogenous LC3-II levels did not change in USP14−/− MEFs in the presence or absence of BafA1 (100 nM).
(E) USP14 inhibition (50 μM IU1 for 6 hr) abrogated the LC3-II accumulation by BafA1 treatment.
(F and G) WT and USP14−/− MEFs were transfected with GFP-LC3 for 24 hr, incubated with 100 μM IU1 for 8 hr, and prepared for the analysis of GFP-LC3 puncta. Representative images (F) and quantification of LC3 puncta (G) of the cells are shown. Values are the mean ± SD of three independent experiments, including a total of ∼1,000 cells.
(H) Electron microscopy of HEK293 cells after treatment with DMSO or 100 μM IU1 for 6 hr. Arrowheads indicate autophagosomes with double membranes, and the scale bars represent 1 μm. N, nucleus.
(I) Quantification of the percentage of cells with the RFP+GFP+ (representing autophagosome)- or RFP+GFP− (autolysosome)-LC3 puncta. HEK293 cells were transfected with control or USP14 siRNA, followed by transient overexpression of RFP-GFP-LC3. Cells with more than 10 puncta were counted, and the data were normalized to total cell numbers (presented as DAPI). Error bars represent the mean ± SD (n = 3).
(J) Same as in (I), except that the experiment was conducted in HEK293 cells with IU1 treatment (100 μM, 8 hr). (Left) The fraction of the autolysosome (RFP-LC3 puncta) was determined and normalized to the merged images with RFP+GFP+ puncta (autophagosome). (Right) The average size of GFP-positive puncta in the presence or absence of IU1 was determined. Bars indicate the mean ± SD of three independent analyses of 10 different images (including >1,000 cells).
(K) Significantly reduced colocalization of overexpressed GFP-labeled LAMP1 with RFP-labeled LC3 puncta in USP14-inhibited cells. HeLa cells stably expressing GFP-LC3 were treated with 50 μM IU1 for 24 hr and immunostained with an anti-LAMP1 antibody. The graphs show the results of three independent experiments where a total of ∼500 cells were analyzed and indicate mean ± SD.
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5. Figure 3
Accelerated UVRAG Degradation through USP14 Inhibition May Lead to the Defects in Autolysosome Formation
(A) UVRAG levels were decreased by a USP14 knockdown. HEK293 cells were transfected with siUSP14 or siCtrl, and the levels of endogenous UVRAG and BECN1 were examined by SDS-PAGE followed by IB.
(B) The UVRAG amount was decreased by USP14 inhibition. HEK293 cells were incubated with vehicle (Veh, 0.1% DMSO) or 50 μM IU1 for 8 hr.
(C) UVRAG was virtually absent in USP14−/− MEFs.
(D) UVRAG levels were elevated after proteasomes were inhibited. HEK293 cells were treated with the indicated concentrations of MG132 for 6 hr.
(E) FLAG-tagged UVRAG (FLAGUVRAG) and/or hemagglutinin (HA)-tagged Ub (HAUb) was transiently overexpressed in HEK293 cells for 24 hr. IU1 (50 μM) or proteasome inhibitors (10 μM MG132, 1 μM PS341) were added 6 hr before lysis. WCEs were subjected to immunoprecipitation with an anti-FLAG antibody, followed by IB with an anti-HA antibody.
(F) UVRAG interacts with USP14. HEK293 cells were transfected with indicated plasmids for 24 hr. Whole-cell extracts (WCEs) were then collected and subjected to co-immunoprecipitation assays. Beclin1 was used as a positive control of UVRAG-interacting proteins.
(G) Transient overexpression of WT FLAGUSP14 (USP14-wt) in HEK293 cells significantly increased cellular UVRAG levels, whereas catalytically inactive FLAGUSP14 (USP14-mut) had little effect. USP14 plasmids were cotransfected with HAUVRAG into HEK293 cells.
(H) Accelerated UVRAG degradation in the presence of IU1. FLAG-tagged UVRAG protein was transiently overexpressed in HEK293 cells, and then chase experiments were carried out at the indicated time points after the addition of 75 μg/mL cycloheximide (CHX) at time zero. The anti-β-actin antibody was used for a control.
(I) Quantification of FLAGUVRAG signals in (H), normalized to those of endogenous β-actin.
(J) Restored BafA1 responsiveness after UVRAG overexpression. USP14−/− MEFs were transfected with plasmids expressing HAUVRAG. After 48 hr, the cells were incubated with 100 nM BafA1 for 4 hr, and endogenous LC3-II amounts were analyzed by SDS-PAGE followed by IB.
(K) Endogenous UVRAG amounts were compared between control cells (parental HEK293) and HEK293-derived stable cells overexpressing FLAG-tagged α3ΔN (α3ΔNflag), which have excessive proteasome activity.
(L) LC3-II accumulation under the influence of BafA1 treatment (100 nM for the indicated periods) was not observed when UVRAG was deficient. HEK293 cells were transfected with siControl or siUVRAG. After 48 hr, the cells were treated with 100 nM BafA1, and then WCEs were prepared and used to examine accumulation of LC3-II by SDS-PAGE followed by IB.
(M) Endogenous USP14 and Ub levels were examined in HCT116 cells and other colon cancer cells. Asterisks denote nonspecific signals.
(N) In HCT116 cells, which lack UVRAG, levels of LC3-II and p62 were not altered by USP14 inhibition. HCT116 cells were treated with DMSO or 50 μM IU1 for 6 hr.
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6. Figure 4
The Compensatory Negative-Feedback Connection between the UPS and Autophagy
(A) HEK293 cells were incubated with an amino-acid-depleted (-AA) medium for the indicated periods. Protein samples were collected and analyzed by SDS-PAGE followed by IB. Accumulated LC3-II after 1- to 4-hr amino-acid starvation represents induced autophagy.
(B) Kinetic measurement of proteasome activity using suc-LLVY-AMC hydrolysis. Autophagy in HEK293 cells was induced through amino-acid (-AA) or glucose (-Glc) starvation for 12 hr in the presence or absence of proteasome inhibitor MG132 (10 μM for 4 hr). Graphs were normalized with WCE concentrations and proteasome activity with MG132. Inserted is the IB for ADRM1 and β-actin, indicating identical proteasome and total protein amounts in the samples.
(C) Increased levels of transiently overexpressed RGS4 under autophagy induction conditions. HEK293 cells were transfected with RGS4-expressing plasmids for 24 hr and then incubated with a normal or starvation medium (-AA or -Glc) for 0, 1, 2, 4, or 8 hr. Control lanes (no RGS4 transfection and MG132 treatment) were removed and indicated with black lines.
(D) Same as in (B), except that WCEs from WT (ATG5+/+) and ATG5−/− MEFs were subject to measurement of proteasome activity.
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7. Figure 5
Inhibition of USP14 Accelerates Degradation of Soluble Forms of MAPT and Delays Its Oligomerization, while Having the Opposite Effects on HTT with Long PolyQ Repeats
(A) MAPT levels increased with USP14 overexpression. MAPT-WT (the longest isoform of human MAPT) or aggregation-prone MAPT-P301L mutant (MAPT-PL) was cotransfected with USP14-WT or USP14-mut into HEK293 cells.
(B) HEK293-Trex-MAPT cells were treated with doxycycline (Dox) (5 ng/mL, 48 hr) and with the indicated concentrations of IU1 for 8 hr. WCEs were prepared in the presence or absence of the reducing reagent β-mercaptoethanol (βME) and were analyzed by SDS-PAGE followed by IB.
(C) Reduced MAPT levels in the fly head after proteasome activation by IU1. Neuron-specific MAPT expression was induced by RU486 supplementation of the diet, in the presence of IU1 or its structural control (c), for 4 days. α-Tubulin served as a loading control.
(D) Representative live images of MAPT oligomerization using the MAPT-BiFC cell line after IU1 treatment (50 μM, 8 hr) in the presence of the phosphatase inhibitor okadaic acid (30 nM, 24 hr).
(E) Quantification of (D). Data indicate the mean ± SD of three independent experiments with more than 1,000 cells.
(F and G) HEK293 cells stably expressing GFP-tagged HTT-Q97 (F) or HTT-Q25 (G) were treated with 10 μM MG132 (6 hr), 100 nM BafA1 (4 hr), or 50 or 100 μM IU1 (6 hr). WCEs were prepared and used for SDS-PAGE followed by IB.
(H) Quantification of the cells with nuclear HTT inclusion. HEK293 cells stably expressing GFP-tagged HTT-Q25, HTT-Q72, or HTT-Q97 with nuclear localization signals were treated with IU1 (10 μM) for 24 hr. Data indicate the mean ± SD of three independent experiments.
(I) Transgenic Drosophila models inducibly expressing HTT-Q18 or -Q152 in neurons under the influence of RU486 were treated with IU1 or its structural control (c) for 7 days.
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8. Figure 6
Illustration of the UPS-Autophagy Negative Feedback through USP14
Schematic representation of the role of USP14 in fluxes of the UPS and autophagy, and their negative-feedback connection. Proteasomes were in a tonic inhibitory state via the catalytic and noncatalytic functions of USP14. Active USP14 also specifically deubiquitinates UVRAG, rescuing it from proteasomal degradation, and this phenomenon may help to maintain the basal level of autophagic flux in the cell. On the contrary, when USP14 is chemically or genetically inactivated, UVRAG is polyubiquitinated and degraded via the 26S proteasome. These events cause impairment of the autophagosome-lysosome fusion step. Induction of autophagy delays the proper UPS flux, although the molecular mediator remains to be determined. Red and blue arrows indicate the states when the proteasome and autophagy are activated, respectively.
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