SUPPLEMENTARY MATERIALS AND METHODS

Patient selection and sample preparation

The study was a retrospective analysis of tissue samples from UCLA’s brain tumor bank acquired from patients who had given IRB-approved consent. Tissue samples were selected based on tumor grade (grade III), established molecular subtype (IDH1 mutant versus wild type tumors), and availability of a sufficient quantity of tumor tissue for proteomics analysis. Tumor tissue was surgically removed from patients, frozen in liquid nitrogen and stored at -80oC. Approximately 20-30 milligrams of frozen tissue were placed in 1.5 ml tubes to which 0.8 ml of ice cold labeling buffer (7 M urea, 2 M thiourea, 4% CHAPS and 20 mM Tris-HCl, pH 8.8) was added. The tubes were then filled with 0.6 ml of 1 mm Zirconia beads by volume (Biospec Products, Inc.). Protein was extracted using a high speed FastPrep FP120 instrument (taking care to avoid sample to sample variation during the protein extraction procedure) at 6.5 m/sec for 15 sec. Extracted proteins samples were sonicated in the Bioruptor (Diagenode Inc.) filled withan ice-cold water for 30 seconds on and 1 minute off for a total of six times at the high setting. Samples were centrifuged at 13,500 rpm for 15 min at 4°C, and the protein supernatants were stored at -80oC.

2D-DIGE analysis and mass spectrometry

Protein extracts were cleaned using a methanol/chloroform method [8] and protein pellets were solubilized in labeling buffer (7 M urea, 2 M thiourea, 4% CHAPS and 20 mM Tris-HCl, pH 8.8). Protein concentration was determined using 2D Quant kit (GE Healthcare). For the difference gel electrophoresis (DIGE) experiments, five wild type tumor samples and five IDH1R132H mutants were used. Protein samples were labeled with the N-hydroxysuccinimidyl ester derivates of Cy2, Cy3, and Cy5 dyes according to the manufactures protocol (GE Healthcare). Pooled standard sample was prepared by combining equal 25 μg amounts of protein from all ten samples and labeled with Cy2. After the labeling, 50 μg of Cy2, Cy3 and Cy5-labeled samples and 300 μg of unlabeled protein mix from all samples were mixed and the rehydration solution was added (7M urea, 2M thiourea, 4% CHAPS, 1% DTT. 0.5% pH 4-7 IPG buffer, 5% glycerol, 10% isopropanol) up to 450 microliters. Samples were applied to 24 cm pH 4-7 IPG strips (GE Healthcare), and rehydrated overnight at room temperature. Isoelectric focusing was performed with the following steps: 200 V (hold) for 2hrs, 500 V (hold) for 2 hrs, 1500 V (gradient) for 2 hrs 30 min, 8000 V (gradient) for 4 hrs, and 8000 V (hold) for 6 hrs 30 min for the total of 77000 Vhrs. IPG strips were equilibrated in a 10 ml of reducing solution (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 1% DTT, 0.002% bromophenol blue) for 15 min and then a second equilibration was performed with 4.5% iodoacetamide in the same solution as above, but without DTT. After reduction and alkylation of proteins, IPG strips were placed on top of the 12.5% SDS polyacrylamide gels and proteins were initially separated at 50V for 2 hrs and then at 1W per gel at 24°C overnight until bromophenol blue dye ran out of gels. The gels were scanned using the Typhoon Trio Variable Mode Imager (GE Healthcare) at 100 micron resolution using a 488 nm laser/520BP40 filter for Cy2, 532 nm laser/580BP30nm filter for Cy3, and a 633 nm laser/670BP30 filter for Cy5. Gel images were cropped using Image Quant software (GE Healthcare) and analyzed using the Decyder 2D Differential Analysis software v. 6.5 (GE Healthcare). Gels were fixed and stained by SyproRuby. After the analysis proteins of interests were selected based on a fold change and Student’s t test values. Sypro-Ruby stained images were re-matched to the DIGE images and protein spots were picked using the Ettan Spot Picker (GE Healthcare) and digested with trypsin using the ProGest protein digestion robotic system (Genomic Solutions). Proteins were identified by MALDI-TOF/TOF mass spectrometry on an Ultraflex MALDI TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) as described [9] and by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a LTQ XL linear ion trap (Thermo Scientific) mass spectrometry equipped with Nano-LC-2D HPLC (Eksigent).

Immunoblotting Analysis

For the immunoblotting experiments, to confirm an equal transfer of proteins from gels to PVDF membranes, 1 μg of protein from all of the samples from control and mutant tumor samples were labeled with Cy5 Dye. Unlabeled 5 μg of the same protein samples were added to each labeled samples. SDS sample loading buffer was added and samples were incubated at 100oC for 5 min and proteins were resolved on a 15% SDS-PAGE with the cathodic running buffer (192 mM Glycine, 25 mM Tris-HCl, 0.1% SDS) containing an additional 0.5% beta-mercaptoethanol (to prevent aggregation of proteins due re-oxidation of cysteine residues). Proteins were transferred to a PVDF membrane by using the Trans-Blot Turbo Blotting system (Bio-Rad). Gels were scanned as above using Cy5 channel before and after transfer of proteins to the PVDF membrane to quantitatively assess protein transfer from gels to membranes.

The membranes were blocked with 5% nonfat dry milk in the wash buffer “WB” (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 1 hr and then washed three times with WB and incubated with primary antibodies made in WB for 3 hrs at room temperature. After several washes, secondary antibodies conjugated to horseradish peroxidase (GE Healthcare, 1:10000) were added and incubated for 1 hr at RT and then the blots were washed and developed using the ECL Plus reagent (GE Healthcare). Fluorescent component of the ECL Plus reaction was detected by scanning the developed blots on the Typhoon 9410 scanner using 457 nm excitation laser and 520BP40 emission filter. Immunoblots were quantified using the Image Quant software (GE Healthcare). The following primary antibodies were used in the immunoblotting analysis: IDH1R132H (Dianova, DIAH091, 1:250), IDH1 (GeneTex 114484, 1:3,000), αB-crystallin (GeneTex, GTX103053, 1:1,000),GFAP (Millipore, MAB3402, 1:50,000), GSTT2 (Abcam, ab118040, 1:500), PEA15 (Thermo Scientific, PA517326, 1:2,000), GLO1 (GeneTex, GTX105792, 1:1,500), PP2A (GeneTex, GTX113523, 1:1,500), αB-crystallin (ThermoScientific, MA5-15383, 1:2,000), Anti-αB-crystallin peptide (163-175) (Millipore, 238702, 1:2,000), GAPDH (GeneTex, GTX627408, 1:5,000).

Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the three IDH1WT(wild type samples 6, 8, 17) and three IDH1R132H(mutant samples 14, 16, 18) tumors using Trizol (Invitrogen, 15596-026) according to manufacturer’s instruction and was further processed using the Fast-Spin column method with RNA Clean and Concentrator-5 (Zymo Research). cDNA was prepared using 0.5μg of RNA from each sample with iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad). All qPCR reactions were performed in a CFX96 instrument (Bio-Rad) using NuPCR Gene Expression Assay (Illumina) containing FAM and HEX dyes for duplex reactions. The following specific primers were used in qPCR for αB-crystallin (forward, 5’-GGTTTCATCTCCAGGGAGTTCCAC-3’and reverse, 5’-CTGTTTCCTTGGTCCATTCACAGTGA-3’), GAPDH (forward, 5’- GTGAAGGTCGGAGTCAACGGATTTGGTC-3’ and reverse, 5’-AGGTCAATGAAGGGGTCATTGATGGCAAC-3’). Each qPCR sample was run in triplicate. GAPDH gene expression was used as an endogenous control gene. qPCRs were performed at the following conditions: 2 min at 95°C, 40 cycles of 15s at 95°C, 30s at 50°C and 30s at 72°C. Relative gene expression of αB-crystallin was calculated using comparative CT method (CT method) [10].

Immunoblot analysis of αB-crystallin protein forms in 2D gel

For immunoblot analysis of different forms of αB-crystallin protein, 5 μg of proteinfrom control and mutant tumor samples were labeled with Cy5 Dye as described above and combined with 20μg of corresponding unlabeled control and mutant protein samples. The volume of samples was brought to 250 microliters with a rehydration solution containing 0.5% 3-11 IPG buffer and 1% DTT and samples were applied to 3-11 NL (non-linear) 13 cm IPG strips and rehydrated overnight. IEFwas performed with the following conditions: 500 V (hold) for 30 min, 1000 V (gradient) for 2 hrs, 8000 V (gradient) for 2 hrs 30 min, 8000 V (gradient) for 45 min for the total of 15080Vhrs. Reduction, alkylation, gel running conditions, transfer of proteins to a PVDF membranes and immunoblotting were the same as described above except proteins were separated on 8-16% SDS gradient gels (Bio-Rad).

Immunohistochemistry

Paraffin embedded tumor sections were incubated overnight at 60°C. Slides were deparaffinized in 3 changes of xylene, rehydrated in a series of ethanol and washed with deionized water. Antigen retrieval was performed by heating sections in Antigen Retrieval buffer (Bionet Medical CB910M) in a Digital Decloaking Chamber (SP1at 120°C for 5 minutes, SP2 at 90°C for 30sec, and SP limit 10°C) and rinsed with two changes of deionized water. Sections were immerged in water and 0.1%TBST for 5 minutes each. Sections were blocked for endogenous peroxidase for 5 minutes in 3% H2O2, rinsed in 3 changes of wash buffer (0.1%TBST), incubated 15 minutes in Background Sniper (BS966L, Biocare Medical), rinsed twice in wash buffer and incubated with primary antibodies, GFAP (BD Pharmingen, 556330, 1:200), IDH1R132H mutant (Dianova, DIA-H09, 1:400), αB-crystallin (ThermoScientific, MA5-15383, 1:500) and GSTT2 (Abcam, ab118040, 1:600). Sections were washed 3 times in wash buffer before applying Mach-4 secondary kit (Biomedical M4U534L). Sections were washed 3 times in wash buffer and stained with NovaRED Chromagen substrate kit (Vector laboratories SK4800). Sections were washed with distilled water, counterstained with hematoxylin for 30 seconds, rinsedwith deionized water. Tacha’s Bluing solution was applied for 1.5 minutes and rinsed with deionized water, dehydrated in series of ethanol and cleared in 3 changes of xylene. Slides were mounted in mounting media and read by a pathologist.

Cell culture

The human wild type IDH1 gene amplification, cloning, mutation and stable expression of Flag-IDH1WT and Flag-IDH1R132H constructs in U251 glioma cell line were performed as described previously [4]. All cells were cultured in Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 units/mL of penicillin/streptomycin at 37°C and 5% CO2 in a 90% humidified tissue incubator. Cell lysates were prepared by sonication in RIPA buffer (Cell Signaling 9806) and protease inhibitor cocktail (Roche 1187358000). Protein concentration was determined by BCA protein assay (Thermo Scientific) and 15μg of protein was loaded onto SDS-PAGE.Proteins were transferred to PVDF membranes and probed with antibodies, as described above.

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