Article
Virus particles were produced in HEK293T cells co-transfected with
Gag-Pol, VSV-G, and RAD23B retrovirus plasmids in six-well plates.
After 12 h of transfection, the medium was replaced and the cells were
cultivated for an additional 24 h. Viral supernatants were then used to
infect RAD23B-KO cells. Puromycin-resistant clones were isolated and
validated by western blotting.
Microscopy
Live-cell imaging experiments were performed on a CV1000 automated
spinning-disk microscope (Yokogawa Electric Corporation) equipped
with a UPLSApo60 × O 1.35NA (Olympus), or on an IX73 inverted fluo-
rescence microscope (Olympus) equipped with an enhanced CSU-X1
spinning disk (Microlens-enhanced dual Nipkow disk confocal scan-
ner) (Yokogawa), a PlanApo 100 × OTIRF 1.45NA (Olympus), and an
Andor Neo sCMOS camera (Andor) or an ORCA-Flash4.0 V3 Digital
CMOS camera (Hamamatsu Photonics). All cells were maintained in
a humidified environment at 37 °C under 5% CO 2. Three-dimensional-
imaging experiments were performed on a Leica TCS SP8 laser-scanning
microscope (Leica Microsystems) with a HC PL APO 100×/1.40 NA oil
CS2 (Leica). In vitro droplet formation of protein samples was moni-
tored on a Fluoview FV3000 confocal laser scanning microscope with
an IX83 fully motorized inverted microscope and a UAPON 100× OTIRF
1.49 NA (Olympus).
Immunofluorescence and time-lapse imaging
Cells were initially plated in 35-mm glass-bottomed dishes (MatTek)
coated with poly-l-lysine. For immunofluorescence, cells were fixed
in 4% paraformaldehyde (Thermo Fisher Scientific) in PBS for 15 min,
and then permeabilized with MeOH (Wako) for 5 min at −20 °C. Before
antibody incubation, cells were blocked with 1% FBS in PBS. The primary
antibodies used were as follows: anti-multi-ubiquitin mouse mono-
clonal FK2 (NBT-MFK003; Nippon Bio-Test Laboratories), anti-multi-
ubiquitin rabbit polyclonal (Z0458; Dako), Lys48-Specific anti-ubiquitin
rabbit monoclonal (05-1307; clone Apu2; Millipore), or Lys63-Specific
anti-ubiquitin rabbit monoclonal (05-1308; clone Apu3; Millipore);
anti-PSMA1 mouse monoclonal antibody^39 ; anti-RAD23B rabbit mono-
clonal (13525; Cell Signaling Technology); anti-VCP mouse monoclonal
(ab11433; Abcam); anti-UBE3A rabbit polyclonal (10344-1-AP; Protein
Tech); anti-RPS2 rabbit polyclonal (ab155961; Abcam); anti-RPS6 rab-
bit monoclonal (2217; Cell Signaling Technology); anti-RPS9 rabbit
polyclonal (18215-1-AP; Protein Tech); anti-RPL4 mouse monoclonal
(sc-100838; Santa Cruz Biotechnology); anti-RPL7A rabbit polyclonal
(2415; Cell Signaling Technology); anti-RPL15 rabbit polyclonal (16740-
1-AP; Protein Tech); anti-RPL29 mouse polyclonal (H00006159-B01P;
Abnova); anti-RPL35 rabbit polyclonal (SAB4500233; Sigma-Aldrich);
anti-rRNA mouse polyclonal (sc-33678; Santa Cruz Biotechnology);
anti-PML mouse monoclonal (sc-966; Santa Cruz Biotechnology); anti-
Coilin mouse monoclonal (ab11822; Abcam); anti-BMI1 rabbit mono-
clonal (6964; Cell Signaling Technology); anti-SC35 mouse monoclonal
(ab11826; Abcam); anti-CENPC guinea pig polyclonal (PD030; MBL,
Medical & Biological Laboratories); and anti-phospho-Histone H2A.X
(Ser139) mouse monoclonal (05-636; Millipore). The following second-
ary antibodies were purchased from Thermo Fisher Scientific: Alexa
Fluor 488-conjugated anti-mouse (A-11029), anti-rabbit (A-11036); Alexa
Fluor 568-conjugated anti-mouse (A-11031), anti-rabbit (A-11036), and
anti-guinea pig (A-11075); and Alexa Fluor 647-conjugated anti-mouse
(A-21236), and anti-rabbit (A-21245). Antibodies were diluted in PBS
with 0.1% FBS. Samples were incubated with antibodies for 1 h at room
temperature. After incubation, cells were treated with DAPI (Thermo
Fisher Scientific) for 15 min and coverslipped (Matsunami) with Slow-
Fade Gold (Thermo Fisher Scientific). For time-lapse experiments, the
medium was replaced with Phenol red-free D-MEM/F12 (Thermo Fisher
Scientific) supplemented with 10% FBS. Cells were incubated for 1 h,
transferred to an incubator microscope (described above), maintained
at 37 °C in 5% CO 2 , and imaged for 1 to 3 h.
Data processing of microscopy images
All image analysis was performed using the Metamorph software
(Molecular Devices). For quantification of proteasome foci, maxi-
mum projections of 16 z-stack images (0.2 μm apart) were manually
segmented with the nucleus (identified by DAPI staining), and foci
number and diameter were analysed using Transfluor. For quantifica-
tion of proteasome foci in time-lapse imaging analysis, the diameter
was calculated from the average foci area per cell, and the median per
view field was averaged. To generate 3D images, we took 30 optical
sections spaced 0.06 μm apart. The image view of the 3D point (xy,
yz, xz) was given by projective transformation. For quantification of
circularity of proteasome foci, images were processed through the
close–open filter and analysed by integrated morphometric analysis.
Circularity was calculated according to the following formula: circular-
ity = 4π(area/perimeter^2 ). For quantification of RPL29 condensates, a
maximum projection of three z-stacks (0.2 μm apart) was manually
segmented with the nucleoplasm, and the number of foci per cell and
the diameter were analysed using integrated morphometric analysis.
The Pearson correlation coefficient (r) was calculated from a scatter
plot of the fluorescence intensities of two proteins.FRAP analysis
In-cell FRAP analysis was performed using a Leica TCS SP8 laser-
scanning microscope equipped with a HC PL APO 100×/1.40 NA oil
CS2. Intracellular assemblies were bleached in a circular 1-μm^2 region
of interest using a 3.25-s pulse of the 405-nm laser line at full power.
Recovery was monitored every 0.65 s for 400 frames. In vitro FRAP was
performed using an Olympus Fluoview FV3000 equipped with a UAPON
100× OTIRF 1.49 NA (Olympus). Droplet assemblies were bleached in a
circular 0.4-μm^2 region of interest using a 0.5-s pulse of the 488-nm laser
line at full power. Recovery was monitored every 0.5 s for 180 frames.
Recovery curves were analysed using Metamorph. Plotting and curve
fitting were carried out in GraphPad Prism 7 (Graphpad Software).Transmission electron microscopy
Samples were fixed with 2% paraformaldehyde and 2% glutaraldehyde
in 0.1 M phosphate buffer (pH 7.4) at 37 °C for 30 min, and then at 4 °C
for 30 min. Afterwards, they were fixed with 2% glutaraldehyde 0.1 M
phosphate buffer (pH 7.4) at 4 °C overnight. The cells were then washed
with the same buffer and post-fixed with 2% osmium tetroxide (OsO 4 )
in the same buffer for 1 h. The samples were then dehydrated for 1 h in a
graded ethanol solution and embedded in a resin (Quetol-812; Nisshin
EM) for 2 days, and polymerized at 60 °C for 48 h. Ultrathin sections
(70-nm thickness) were made with a diamond knife on a Leica Ultra-
cut UCT ultramicrotome (Leica), and then mounted on copper grids.
Sections were stained with 2% uranyl acetate and lead stain solution
(Merck) and visualized on JEM-1400Plus electron microscope ( JEOL) at
an acceleration voltage of 100 kV. Digital images (3,296 × 2,472 pixels)
were acquired with an EM-14830RUBY2 camera ( JEOL).Correlative light electron microscopy
Image characteristics (pixel size, lateral resolution, and axial resolu-
tion) of fluorescence emission and electron density were merged using
ec-CLEM on the open-source software platform Icy^40 (Institut Pasteur).
For alignment, this algorithm relies on manual identification of match-
ing landmarks in the cell in single-section images, and it operates with
an accuracy determined by the degree of sample distortion caused by
the intermediate sample preparation step.Immunoprecipitation
Cells were collected and lysed in buffer A (50 mM Tris-HCl, pH 7.5, 100 mM
NaCl, 10% glycerol, 10 mM iodoacetamide) containing 0.2% NP-40
and complete protease inhibitor cocktail (Roche, EDTA-free). After
standing on ice for 30 min the lysate was sonicated on a Handy Sonic