1. Proteasome Activation by Small Molecules
Yves Leestemaker, Annemieke de Jong, Katharina F. Witting, Renske Penning, Karianne Schuurman, Boris Rodenko, Esther A. Zaal, Bert van de Kooij, Stefan Laufer, Albert J.R. Heck, Jannie Borst, Wiep Scheper, Celia R. Berkers, Huib Ovaa 
Cell Chemical Biology 
Volume 24, Issue 6, Pages e7 (June 2017)
DOI: /j.chembiol
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2. Figure 1
Identification of Small-Molecule Proteasome Activators Using a Proteasome Activity Probe
(A and D) In-gel fluorescence scan showing representative proteasome activity profiles in (A) MelJuSo cell lysates treated with MG132 or (combinations of) ADP, K+, or ATP-γS or (D) MelJuSo cells after a 16 hr incubation with 5 μM compound or 750 nM MG132. Bars represent the quantified probe signal.
(B) Rate of suc-LLVY-AMC conversion in MelJuSo cell lysates treated with MG132 or (combinations of) ADP, K+, or ATP-γ. Error bars represent SD of three independent experiments.
(C) Structures of small-molecule compounds that increase proteasome activity.
(E) Intracellular fluorescence intensities in MelJuSo cells after a 16 hr incubation with 5 μM of the indicated compounds, followed by incubation with either probe 1 or reduced probe 1 (termed reactive probe and control probe, respectively). All signals were normalized to intensities obtained in DMSO-treated control (CTR) cells. Error bars represent SD of three independent experiments.
(F) The rate of suc-LLVY-AMC conversion in MelJuSo cells treated with 5 μM of the indicated compounds. The conversion rates in DMSO-treated control (CTR) cells and epoxomicin-treated cells (Epox) were set to 1 and 0, respectively. Error bars represent SD of three independent experiments. Statistical significance: ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ See also Figures S1 and S2.
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3. Figure 2
Chemical and Genetic Inhibition of p38 MAPK Increase Intracellular Proteasome Activity
(A) Structures of p38 MAPK active-site inhibitors PD169316, SB202190, SB203580, and p38 MAPK allosteric inhibitor skepinone-L.
(B) Intracellular fluorescence intensities in MelJuSo cells, treated with probe 1 after a 16 hr incubation period with increasing concentrations of the indicated p38 MAPK inhibitors. All signal intensities were normalized to intensities obtained in DMSO-treated control (CTR) cells. Error bars represent SD of three independent experiments.
(C) In-gel fluorescence scan (top) showing representative proteasome labeling profiles in MelJuSo cells after a 16 hr incubation period with 5 μM of the indicated compounds or 750 nM MG132. Bars represent the quantified probe signal. Immunoblot analysis of the levels of the indicated proteins (bottom). GADPH levels were used as a loading control.
(D) The rate of suc-LLVY-AMC conversion in MelJuSo cells treated with 5 μM of the indicated compounds. The conversion rates in DMSO-treated control (CTR) cells and epoxomicin-treated cells were set to 1 and 0, respectively. Error bars represent SD of three independent experiments. Statistical significance: ns, not significant, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤
(E) Intracellular fluorescence intensities in MelJuSo cells, treated with probe 1 after 72 hr transfection with siRNAs targeting the indicated proteins or control siRNA (siCTR). All signal intensities were normalized to intensities obtained in non-transfected (NT) cells. Error bars represent SD of three independent experiments.
(F) In-gel fluorescence scan (top) showing representative proteasome labeling profiles in MelJuSo cells after 72 hr transfection with either control siRNA or siRNA targeting p38α MAPK. Bars represent the quantified probe signal. Immunoblot analysis of the levels of the indicated proteins (bottom). GADPH levels were used as a loading control.
(G) Intracellular fluorescence intensities in MelJuSo cells, treated with probe 1 after 72 hr transfection with siRNAs targeting the indicated proteins or control siRNA (siCTR), followed by a 16 hr incubation with PD or DMSO. All signal intensities were normalized to intensities obtained in NT cells. Error bars represent SD of three independent experiments.
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4. Figure 3 Involvement of p38 MAPK Pathway in Proteasome Modulation
(A) Normalized intracellular fluorescence intensities in probe 1-treated MelJuSo cells after 72 hr transfection with siRNAs targeting the indicated proteins in the p38 MAPK pathway. The color of the box indicates fold activation of siRNA over control siRNA.
(B) Rate of suc-LLVY-AMC conversion in MelJuSo cells after 72 hr transfection with siRNA targeting the indicated proteins. The conversion rates in cells transfected with control siRNA (siCTR) and epoxomicin-treated cells were set to 1 and 0, respectively. Error bars represent SD of three independent experiments. Statistical significance: ns, not significant, *p ≤ 0.05, and **p ≤ 0.01.
(C) In-gel fluorescence scan (top) showing representative proteasome labeling profiles in MelJuSo cells after 72 hr transfection with either control siRNA or siRNA targeting the indicated proteins. Bars represent the quantified probe signal. Immunoblot analysis (bottom) showing the expression levels of the indicated proteins. GAPDH levels were used as a loading control. See also Figures S3 and S4, Tables S1 and S2.
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5. Figure 4
PD and MK2 Inhibitor III Activate the Proteasome through Distinct Mechanisms
(A) The structure of MK2 inhibitor III.
(B) In-gel fluorescence scan (top) showing representative proteasome labeling profiles of purified proteasome preparations, obtained from HTBH-Rpn11-HEK293T cells after a 16 hr incubation period with 5 μM of the indicated compounds or after 72 hr transfection with siRNA targeting p38 MAPKα. Bars represent the quantified probe signal. Immunoblot analysis (bottom) of the levels of the indicated proteins. GAPDH levels were used as a loading control.
(C) Suc-LLVY-AMC conversion by purified proteasome preparations, obtained from HTBH-Rpn11-HEK293T cells after a 16 hr incubation period with 5 μM of the indicated proteasome activators or 750 nM MG132 (top). Quantification of substrate turnover (bottom). Error bars represent SD of three independent experiments. Statistical significance: *p ≤ 0.05, **p ≤ 0.01, ***p ≤
(D) Intracellular fluorescence intensities in probe 1-treated MelJuSo cells after a 16 hr incubation period with the indicated (combinations of) p38 MAPK inhibitors. All signals were normalized to intensities obtained in DMSO-treated control (CTR) cells. Error bars represent SD of three independent experiments.
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6. Figure 5
p38 MAPK Inhibition Increases Protein Clearance by the Proteasome
(A) Suc-LLVY-AMC conversion in lysates obtained from MelJuSo cells after 72 hr transfection with siRNAs targeting p38f MAPK. Lysates were subsequently supplemented with a recombinant p38αD176A/F372S protein or its inhibitor PD or a combination of both, before AMC fluorescence measurements were performed. Error bars represent SD of three independent experiments. Statistical significance: ***p ≤
(B) HA-GFP levels in MelJuSo cells treated with 5 μM of the indicated p38 MAPK inhibitors or epoxomicin. All signals were normalized to intensities obtained in DMSO-treated control (CTR) cells. Error bars represent SD of three independent experiments. Statistical significance: *p ≤ 0.05, **p ≤ 0.01, ***p ≤
(C) Immunoblot analysis showing the levels of the indicated proteins in HeLa cells that were preincubated with 5 μM PD169316, followed by a 6 hr treatment with 1 μM dBET1. Bars represent the quantified BRD4/CRBN ratio. Three independent experiments were performed.
(D) α-Synuclein-yellow fluorescent protein (YFP) levels (top), BFP levels (middle) and cell survival (bottom) in MelJuSo cells transfected with α-synuclein-split YFP and BFP and treated with 5 μM of the indicated p38 MAPK inhibitors. All signals were normalized to intensities obtained in DMSO-treated control (CTR) cells. NT, non-transfected. Error bars represent SD of three independent experiments. Statistical significance: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ and ****p ≤
(E) In-gel fluorescence scan (top) showing representative proteasome labeling profiles in primary mouse neurons after a 16 hr treatment with the indicated concentrations of PD or MG132. Immunoblot analysis (bottom) showing the endogenous α-synuclein, β5 and GAPDH expression levels. GAPDH levels were used as a loading control.
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