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Supplementary Materials and Methods
Cell culture and cell viability assay. In MTT assay, cells were seeded in 96-well
plates at a density of 2×104 cells/ml and incubated overnight. Then, the media were
changed into fresh media containing various amounts of celastrol for 48 h. At the end
of the incubation, 20 μl of the dye (3, [4,5-dimethylthiazol-2-yl-] diphenyltetrazolium
bromide, 5 mg/ml), MTT, was added to each well and the plates were incubated for 3
h at 37℃. Then, 100 μl of lysis buffer (20% sodium dodecyl sulfate [SDS] in 50%
N,N-dimethylformamide, containing 0.5% [v:v] 80% acetic acid and 0.4% [v:v] 1N
HCL) was added to each well and incubated overnight (16 h). Cell viability was
evaluated by measuring the mitochondrial-dependent conversion of the yellow
tetrazolium salt MTT to purple formazan crystals by metabolic active cells. The
optical density (proportional to the number of live cells) was assessed with a
Microplate Reader Bio-Rad 550 at 570 nm. For Trypan blue exclusion assay, cells
were seeded in 6-well plates at a density of 1×105 cells/ml and incubated overnight
before celastrol treatment. At the end of celastrol treatment, both the adherent and
detached cells were collected and stained with trypan blue dye for 5 min at room
temperature. The cell suspension was then applied to Invitrogen CountessTM
Automated Cell Counter and cells stained with trypan blue were considered as dead
cells.
Preparation of samples of total cellular proteins and samples of mitochondria
and cytoplasm fractions. For sample preparation of total cellular protein, cells were
washed three times with ice-cold PBS and then scraped off with a cell scraper. After
centrifugation, cell pellets were dissolved in lysis buffer (7 M urea, 2 M thiourea, 2%
CHAPS, 1% DTT, 0.8% Pharmalyte, and protease inhibitor). Homogenization of the
cells was achieved by ultrasonication (10 strokes, low amplitude) on ice. After
homogenization, the lysed cells were centrifuged at 15,000×g for 30 min at 4 °C, and
the supernatant containing the solubilized proteins was used directly or stored at
-80 °C. Protein samples from at least three independent experiments were collected
for 2-DE analysis and Western blotting assay.
For preparation of mitochondria and cytoplasm fractions, a Cell Mitochondria
Isolation kit (Beyotime, China) was used. Cytoplasmic and mitochondrial proteins
were extracted from cells according to the manufacturer’s instructions. Briefly, cells
were collected using a cell scraper and then washed with pre-cooled PBS buffer. After
centrifugation, cells (approximately 2x107 cells) were resuspended in 1 mL
mitochondria isolation buffer. After incubate for 10 min on ice, cells were
homogenized using a MP FastPrep®-24 Homogenizer (4 m/s, 15s, 3 times). After
centrifugation, the pellet (mitochondria fraction) and the supernatant (cytoplasm
fraction) could be used to extract protein using the method similar to extraction of
total cellular protein.
Western blotting assay. The primary antibodies used were rabbit anti-caspase-3 (Cat.
#9665, Cell Signaling Technology) antibody (1:600), rabbit anti-caspase-9 (Cat.
#9502, Cell Signaling Technology) antibody (1:800), rabbit anti-PARP (Cat. #9532,
Cell Signaling Technology) antibody (1:1000), rabbit anti-XIAP (Cat. #2045, Cell
Signaling Technology) antibody (1:1000), mouse anti-TOM22 (Cat. #T6319, clone
1C9-2, Sigma) monoclonal antibody (1:1000), rabbit anti-Bax (Cat. #2774, Cell
Signaling Technology) antibody
(1:800), rabbit anti-ERP29 (Cat. #ab137670,
Abcam) antibody (1:2000), rabbit anti-Bip (Cat. #3177, Cell Signaling Technology)
antibody
(1:1000), rabbit anti-IRE1 (Cat. #3294, Cell Signaling Technology)
antibody
(1:1000), rabbit anti-PERK (Cat. #3192, Cell Signaling Technology)
antibody
(1:1000), rabbit anti-p-JNK (Cat. #4668, Cell Signaling Technology)
antibody (1:1000), rabbit anti-caspase-4 (Cat. #4450, Cell Signaling Technology)
antibody (1:800), rabbit anti-phospho-eIF2α (Ser51) (Cat. #9721, Cell Signaling
Technology) antibody (1:400), mouse anti-XBP1s (Cat. #MAB4257, Clone 525904,
R&D) monoclonal antibody(1:1000), rabbit anti-p-IRE1 alpha (Cat. #NB100-2323,
NOVUS) monoclonal antibody (1:800), rabbit anti-GSK3β (Cat. #9315, Cell
Signaling Technology) antibody (1:1000), rabbit anti-p(ser9)-GSK3β (Cat. #9336,
Cell Signaling Technology) antibody (1:1000), and rabbit anti-actin (Cat. #4970, Cell
Signaling Technology) antibody (1:1000).
Assay of activities of cellular proteasome. Catalytic activities of the catalytic
subunits of proteasome (β1, β2, β5) were assayed by adding whole cell extract
(containing 50 μg protein) to 100 μl of assay buffer (20 mM Tris-HCl, pH 8.0, 1 mM
adenosine triphosphate, 2 mM MgCl2) containing 50 μM fluorogenic peptide
substrates (Thermo Fisher Scientific, Rockford, IL, U.S.A) such as Z-LLE-AMC
(7-amido-4-methyl-coumarin) for β1 subunit, Z-ARR-AMC for β2 subunit or
Suc-LLVY-AMC for β5 subunit, respectively. The hydrolase activity of the catalytic
subunits could release the fluorogenic AMC component from the peptide substrates
and the AMC release was measured after 30 minute incubation using a Microplate
Reader Bio-Rad 550 with excitation and emission wavelengths of 360 and 480 nm,
respectively.
Expression of recombinant proteasome catalytic subunit β1. Recombinant human
proteasome catalytic subunit β1 was expressed as a His-fusion protein using
Escherichia coli, and purified by affinity chromatography. Briefly, the PSMB6
full-long cDNA incorporating EcoR I and Xho I sites was synthesized by Life
Technologies Corporation (Shanghai, China). The EcoR I - Xho I fragment of the
PSMB6 gene was then subcloned into pET-28a expression vector (Merk) and
transformed into E. coli BL21(DE3) strain (Merck Drugs & Biotechnology,
Darmstadt, Germany). The BL21 transformants were cultivated in LB medium with
ampicillin to an A600 nm of 0.8 at 37℃, induced by adding isopropyl
b-D-thiogalactopyranoside to a final concentration of 1 mM, and then incubated
overnight at 20℃. The fusion protein, containing His-tag, was isolated from bacterial
lysates by Ni2+-chelation affinity chromatography using the Profinia protein
purification system (Bio-Rad). This purification system improved purity of fusion
protein to more than 95% (SDS-PAGE analysis), and ensured the integrity of
polypeptide.
2-DE analysis and MALDI-TOF MS/MS. Briefly, 150 mg cellular total protein
sample was applied for IEF using the ReadyStrip IPG Strips (17 cm, pH 4-7). The IEF
was conducted using the following protocol: 250V, linear, 30 min; 1000V, rapid, 1 h;
10000V, linear, 5 h; 10000V, rapid, 60,000 Vh. After IEF, the IPG strips were
equilibrated and then directly applied on to 12% homogeneous SDS-PAGE gels for
electrophoresis using a PROTEIN II xi Cell system (Bio-Rad). The gels were silver
stained using Silver Stain Plus kit reagents (Bio-Rad). Stained gels were scanned with
a Densitometer GS-800 (Bio-Rad) and the protein spots were quantified using the
PDQuest 7.4.1 software (Bio-Rad). The individual protein spot quantity was
normalized as follows: the raw quantity of each spot in a member gel was divided by
the total quantity of the valid spots in the gel, and normalized spot intensities were
expressed in ppm. Comparisons were made between gel images obtained from
celastrol-treated group and control group. Quantitative analysis was performed using
the Student’s t-test between the two groups. The significantly differentially expressed
protein spots (p<0.05) with 1.5 fold or more increased or decreased intensity were
selected, punched out of the gels and subjected to further identification using
MALDI-TOF MS/MS. To ensure reproducibility, paired (control and celastrol-treated)
protein samples from 3 independent experiments were analyzed by 2-DE and
triplicate electrophoreses were performed for each pair of protein samples.
For MS identification, the protein spots were destained, equilibrated, dehydrated
and then dried. Spots were then rehydrated in trypsin solution and incubated overnight
at 37℃. The supernatant containing extracted peptides was directly applied onto the
sample plate with equal amounts of MALDI matrix. MALDI-TOF MS/MS analysis
was conducted using the same parameters as described in detail in our previous report
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with an Applied Biosystems 4700 Proteomics Analyzer (Framingham, MA). Briefly,
the probability-based score had to be more than 64 when submitting PMF data to the
database and be more than 30 for individual peptide ions when submitting peptide
sequence spectra, assuming that the observed match is significant (P<0.05).
PCR analysis. For PCR analysis, total RNA samples were isolated from control and
celastrol-treated cells using the Total RNA isolation system (Life Technologies, Grand
Island, NY, USA). Equal amounts of the RNA isolated from celastrol-treated cells or
control cells were transcribed into cDNA with PrimeScript® RT Master Mix (TaKaRa
Bio Group, Japan) and analyzed by RT-PCR using SYBR® Premix Ex Taq™ Kit
(TaKaRa). The primer pairs for the genes including Tom5, Tom6, Tom7, Tom20,
Tom22, Tom70, BAD, BID, BIK, BMF, HRK, NOXA, PUMA and Actin (as internal
control) were showed in Supplementary Table S5. The thermal program included a 10
min incubation at 95℃ to activate the FastStart Taq polymerase followed by 40 cycles
of 95℃ for 10 s, 58℃ for 10 s and 72℃ for 20 s. The fluorescence readings were
recorded after each 72℃ step. Dissociation curves were performed after each PCR
run to ensure that a single PCR product had been amplified. RT-PCR analysis using
RNA from three independent experiments were conducted.
Transfection of GFP-Bax. For transiently GFP-Bax transfection of HeLa cells, cells
were seeded into six-well plates in medium without antibiotics to achieve a monolayer
density of 60% to 70%. Then, for each well, 1.2 μg of GFP-Bax plasmid DNA diluted
in 50 μl of medium without serum was mixed with 2 μl of Lipofectamine™ 2000
(Life Technologies) diluted in 50 μl OptiMEM® I Medium (Life Technologies) and
then incubated at room temperature for 20 min to allow DNA-Lipofectamine™ 2000
complexes to form. Transfection was conducted by incubating cells with the
DNA-Lipofectamine™ 2000 complex solution for 4 h at 37°C in a CO2 incubator.
iTRAQ-based LC-MS analysis of protein expression profile of ER/microsomal
vesicles-enriched fraction of cells with or without celastrol treatment. Briefly,
cells were collected, washed with PBS, and resuspended in MS buffer (5 mM
Tris-HCl (pH 7.5), 210 mM mannitol, 70mM sucrose, 1 mM EDTA ) supplemented
with protease inhibitors (Thermo Fisher Scientific, Pittsburgh, USA). The cells were
lysed by homogenization and the cell lysate was centrifuged at 80×g for 10 min at
4°C. Then, the supernatants were further centrifuged at 14000×g for 20 min at 4°C.
The supernatants were then centrifuged at 350000×g for 20 min at 4°C and the pellets
(ER/microsomal vesicles-enriched fraction) were resuspended in MS buffer. The
protein concentrations of the ER/microsomal vesicles-enriched fractions were
measured by Bradford assay (Bio-Rad).
An aliquot of ER/microsomal vesicles-enriched fraction sample containing 100
µg protein was reduced and blocked as suggested by the manufacturers. The proteins
were digested overnight with Trypsin (10:1) at 37°C. The resulting peptides were
subsequently labeled using iTRAQ 4-plex kit
(AB SCIEX) according to the
manufacturer's instruction. To ensure reproducibility and exclude labeling bias, each
pair of protein samples (control and celastrol-treated) were labeled with two kinds of
labeling strategy. The digested samples were labeled with different iTRAQ tags as
follows: control, iTRAQ 114 or 115; celastrol-treated, iTRAQ 116 or 117. After
labeling, the samples were pooled and dried.
For sample fraction, the iTRAQ-labeled samples were reconstituted using strong
cation exchange (SCX) buffer A (10mM monobasic potassium phosphate, pH 2.75,
25% acetonitrile) and the pH values of the samples was adjusted to 2.5-3 with
phosphoric acid. Samples were separated by SCX HPLC using a Poly SULFOETHYL
A column (100×4.6mm, 5 mm, 300 Å, PolyLC, Columbia, MD, USA) on a Lab
Alliance HPLC system. After a 20 minute wash at 0.5 ml/min in 100% buffer A, an 80
minute linear gradient from 0 to 100%v/v buffer B (same as buffer A, with the
addition of 0.5M potassium chloride) was run and fractions were collected every
minute based on UV (220 nm) absorbance. Afterwards, the samples were completely
dried in a vacuum centrifuge. In LC-MS analysis, the samples were reconstituted in
0.1% formic acid.
The nano-ESI MS/MS analysis was carried out using an Eksigent ultra+nano flex
cHiPLC system coupled to a quadrupole time-of-flight TripleTOF 5600 mass
spectrometer (AB SCIEX). Each sample (5 μl) was loaded onto a trap column
(Eksigent ChromXP C18-ClP, 200 μm×0.5 mm, 3 μm, 120 Å，USA) at a flow rate of
4 μl/min for 5 min. Peptide separation was carried out on a C18 column (Eksigent
ChromXP C18-Cl, 75 μm×15cm, 3 μm, 120 Å, USA) at a flow rate of 300 nl/min.
Peptides with iTRAQ labels were separated using a 125 minute gradient ranging from
5% to 50% mobile phase B (mobile phase A: 2% acetonitrile, 0.1% formic acid;
mobile phase B: 98% acetonitrile, 0.1% formic acid). The MS analysis was performed
on the TripleTOF 5600 system in information dependent acquisition mode. The
setting parameters were: ion spray voltage: 2300 V; curtain gas: 30; nebuliser gas: 30
and heated interface: 150°C. Tandem mass spectrawere acquired over the mass range
m/z 100–1500 using rolling collision energy for optimum peptide fragmentation.
The mass spectrometry data files were processed by ProteinPilot 4.0 (AB SCIEX)
using the Paragon algorithm. The mass spectrometry data were searched against all
the protein sequence Homo sapiens of Uniprot_sprot_201105 database.For
ProteinPilot Paragon, methyl methane thiosulfonate was selected as the cysteine
modification agent, trypsin as the digestion enzyme, ‘biological modifications’ were
selected as the ‘ID focus’ and a ‘Thorough ID Search Effort’ was selected. False
discovery rate analysis was performed using reversed protein sequences and was used
to calculate the number of false positive proteins expected at a 95% confidence level.
Peptides that passed a 1% FDR threshold were considered for protein identification
and quantification. To ensure reproducibility, the average value of the reporter ion
intensities of two technical replicates was used for relative quantification. The relative
amount of a peptide in each sample was calculated by dividing the peak areas
observed at 116.1, and 117.1 m/z by that observed at 114.1, 115.1m/z. The calculated
peak area ratios were corrected for overlapping isotopic contributions and used to
estimate the relative abundances of a particular peptide. For proteins with two or more
qualified peptide matches, four average peak area ratios (designated as 116/114,
117/114, 116/115, 117/115) were calculated using the peak area ratios of the peptides
originating from the same protein. In the present study, only proteins whose four peak
area ratios were all >1.5 or <0.67 between celastrol-treated cells and control cells
were accepted as differentially expressed proteins.