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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 53 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.