Methods
No statistical methods were used to predetermine sample size. The
experiments were not randomized. The investigators were not blinded
to allocation during experiments and outcome assessment.
Cell culture
HCT116 cells (human colorectal carcinoma, CCL-247) and hTERT RPE-1
cells (human telomerase-immortalized retinal pigmented epithelial,
CRL-4000), both obtained from ATCC (Manassas) were maintained at
37 °C in Dulbecco’s modified Eagle’s medium (Sigma) supplemented
with 10% (v/v) fetal bovine serum (FBS) (Biowest), 1 mM sodium pyru-
vate (Thermo Fisher Scientific), and nonessential amino acids (Thermo
Fisher Scientific) in an incubator with a 5% CO 2 atmosphere. E14TG2a
mouse ES cells purchased from ATCC (CRL-1821) were cultured in Dul-
becco’s modified Eagle’s medium (Sigma) supplemented with 10% (v/v)
Embryonic Stem-Cell FBS (Thermo Fisher Scientific), 1 mM sodium
pyruvate, nonessential amino acids, 0.1 mM 2-ME (Sigma), 15% (v/v)
FBS-free medium supplement (Thermo Fisher Scientific), and 10^3 U ml−1
unit ESGRO Leukaemia Inhibitory Factor (Merck).
Inhibitors and reagents
Formation of proteasome foci was induced by NaCl (Wako),
sucrose (Wako) or glucose (Wako) at the indicated concentrations.
Me4BodipyFL-Ahx3Leu3VS fluorescent proteasome probe^31 (UbiQ
Bio) was used to detect endogenous proteasome activity. PSMB2-
FusionRedKI/KI cells were treated with 1 μM Me4BodipyFL-Ahx3Leu3VS
for 1 h and washed with PBS, after which formation of proteasome
foci was induced with 0.2 M sucrose. Ammonium acetate (Wako) and
1,6-hexanediol (Sigma) were used to distinguish liquid-like and solid-
like states of proteasome foci in living cells. Hexanediol (1%) and 0.1 M
ammonium acetate were added 2 min after induction of foci formation
by 0.2 M sucrose in PSMB2-eGFPKI/KI cells. Because hexanediol can cause
various deleterious effects on cells, treatment with this reagent was
minimized^32. The inhibitors used in this study were as follows: bort-
ezomib (LC Laboratories), MG-132 (Peptide Institute), b-AP15^33 (LifeSen-
sors), MLN-7243^34 (Active Biochem), NMS-873^35 (Selleck Chemicals),
DBeQ (Sigma) and CX-5461 (ChemScene).
MTS assay for cell viability
Cells treated with different concentration of DBeQ for 4 h, and cell
viability was analysed by CellTiter 96 AQueous One Solution Cell Pro-
liferation Assay kit (Promega). Absorbance at 490 nm was measured
on an EnSpire 2300 multimode plate reader (PerkinElmer).
Plasmids and plasmid transfection
Transfections of HCT116 cells with the indicated plasmids were per-
formed using Lipofectamine 2000 (Thermo Fisher Scientific). Human
RPL7A and RPS2 were cloned into pcDNA3-eGFP (Addgene 13031) with a
GGGS linker sequence and 3×Flag to yield pCDNA3-RPL7A-eGFP-3×Flag
and pcDNA3-RPS2-eGFP-3×Flag, respectively. To generate pcDNA3-
RAD23A-FusionRed-3×Flag, human RAD23A was cloned with a GGGS
linker sequence and 3×Flag into pcDNA3-eGFP in which the eGFP gene
was replaced with FusionRed (Evrogen, JSC). To construct donor vec-
tors for TALEN knock-in (KI), ~1,000 bp upstream of the target site,
including the last exon without the stop codon, a linker sequence (GGGS
linker), eGFP or FusionRed, 3×Flag tag, SV40 polyA, selection marker,
and ~1,000 bp of downstream sequence were tandemly flanked by PCR
and inserted into pBlueScript (Stratagene).
RNA interference
siRNA oligos were obtained from ON-TARGETplus SMARTpool (Dhar-
macon). The target sequences are as follows: RAD23A#1, GCTCTGAGT
ATGAGACGAT; RAD23A#2, GAAGATAGAAGCTGAGAAG; RAD23A#3,
GATCTTGAGTGACGATGTC; RAD23A#4, GAAGAACTTTGTGGTCGTC;
RAD23B#1, GCAGATAGGTCGAGAGAAT; RAD23B#2, GAACGAGA
GCAAGTAATTG; RAD23B#3, GAAGTGGTCATATGAACTA; RAD23B#4,
CAACAACCCTGACAGAGCA; UBQLN1#1, CATCAACTCCTAATAGTAA;
UBQLN1#2, GTACTACTGCGCCAAATTT; UBQLN1#3, AGACAAACGTT
GGAACTTG; UBQLN1#4, CTGAGTAGCTTGGGTTTGA; UBQLN2#1,
TAAGGAAGCGATTTCGAAA; UBQLN2#2, CTGAATAGCCCGCTGTTTA;
UBQLN2#3, CCAAACCGATCAGCTAGTG; UBQLN2#4, GACATTAG
CCACTGAAGCA; UBQLN4#1, GCTGAGAATATGACGGCAA; UBQLN4#2,
CCAATGAAGCTAAGCGCCA; UBQLN4#3, GAGGAGGGAATGAGATTAT;
UBQLN4#4, CCAACCAATGCTAGAATTT; PML#1, GGAAAGATGCAGC
TGTATC; PML#2, GAGCTCAAGTGCGACATCA; PML#3, GGACA
TGCACGGTTTCCTG; PML#4, GCAACCAGTCGGTGCGTGA; XPC#1,
GCAAATGGCTTCTATCGAA; XPC#2, TGAAATATGAGGCCATCTA; XPC#3,
GAGAAGTACCCTACAAGAT; XPC#4, GGAGGGCGATGAAACGTTT. For
non-targeting control siRNA, we used ON-TARGETplus non-targeted
siRNA#4 (Dharmacon). siRNAs were transfected into cells using Lipo-
fectamine RNAiMAX (Thermo Fisher Scientific). After 24 h of transfec-
tion, the medium was replaced, and the cells were grown for an addition
24 h before analysis.Generation of KI cell lines
PSMB2-eGFP-3×Flag- or PSMB2-FusionRed-KI HCT116 cells were gen-
erated by TALEN KI using the Platinum Gate TALEN Kit, a gift from T.
Yamamoto (Addgene# 1000000043)^36. TALEN-targeting sequences
(PSMB2-1: TTTCCTTCCCCAAACAGGGCT; PSMB2-2: TTCCCTGGCA
AGTGGGAGGGA) at the last exon of PSMB2 were cloned into ptCMV-
153/47. The assembled TALEN plasmids and a donor vector pcDNA3-
PSMB2-linker-eGFP-3×Flag (Puro) or pcDNA3-PSMB2-linker-FusionRed
(Puro) were co-transfected into HCT116 cells. Puromycin-resistant
clones were isolated and validated by western blot and DNA sequencing.
PSMD6-eGFP-3×Flag-KI HCT116 cells were generated using the TALE
nuclease (TALEN) Kit^37 , a gift from F. Zhang (Addgene 1000000019).
TALEN-targeting sequences (PSMD6-1: TCCAGAGTAATTAATATGTA;
PSMD6-2: TCCAGAGTAATTAATATGTA) at the last exon of PSMD6
were cloned into pTALEN_v2. The assembled TALEN plasmids and the
donor vector, pBlueScript-PSMD6-linker-eGFP-3×Flag (Puro), were
co-transfected into HCT116 cells, and puromycin-resistant clones were
isolated and validated. eGFP-ubiquitin, RPL29-eGFP, RPL15-eGFP or
RPS2-eGFP was knocked in to the AAVS1 locus of HCT116 cells using
the TALEN Kit using TALEN-targeting sequences AAVS1-1 (TGTCC
CCTCCACCCCACA) and AAVS1-2 (TTTCTGTCACCAATCCTG). The
TALEN plasmids and a donor vector pBlueScript-EF1pr-EGFP-
ubiquitin (Neo), pBlueScript-EF1pr-RPL15-linker-eGFP-3×Flag (Neo),
pBlueScript-EF1pr-RPL29-linker-eGFP-3×Flag (Neo) or pBlueScript-
EF1pr-RPS2-linker-eGFP-3×Flag (Neo) were co-transfected into HCT116
cells. G418-resistant clones were isolated and validated by immunoblot
and DNA sequencing.Generation of UBE3A-KO and RAD23B-KO cells
A CRISPR guide sequence targeting exon 3 of UBE3A or exon 5 of RAD23B
was cloned into pSpCas9(BB)-2A-Puro (PX459) v.2.0^38 , a gift from F.
Zhang (Addgene 62988). Sequences were as follows: UBE3A-1: CA
CCGCTTACCTTGAGAACTCGAA; UBE3A-2: AAACTTCGAGTTCTCA
AGGTAAGC; RAD23B-1: CACCGAAGATGCAACGAGTGCACT; RAD23B-2:
AAACAGTGCACTCGTTGCATCTTC. Puromycin-resistant clones were
isolated and validated by western blotting and DNA sequencing.Generation of RAD23B add-back cell lines
RAD23B add-back cell lines were generated using a retroviral system.
For expression of human RAD23B, RAD23B mutants, L8A and L225A/
L401A, were generated by PCR mutagenesis using the QuikChange
mutagenesis kit (Agilent Technologies) and cloned into pMX-Puro retro-
viral expression vector (Cell Biolabs) to yield pMX-puro-RAD23B(WT)-
FusionRed-3×Flag, pMX-puro-RAD23B(L8A)-FusionRed-3×Flag, and
pMX-puro-RAD23B(L225A/L401A)-FusionRed-3×Flag, respectively.