Maintaining Normal Cellular Growth Biology Essay

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c-Myc is a proto-oncogenic transcription factor. It is now established beyond doubt that the rapid turnover of c-Myc protein mediated by the ubiquitin/proteasome system (UPS) is critical for maintaining normal cellular growth. In this study we report a new E3 ligase for c-Myc, the carboxyl terminus of HSP70 interacting protein or CHIP which is a chaperone associated U box containing E3 ligase. CHIP interacts with c-Myc and leads to its ubiquitination and subsequent proteasome mediated degradation. Over-expression of CHIP could accelerate the turnover rate of c-Myc. Conversely, depletion of CHIP by RNAi stabilizes endogenous c-Myc. The interaction between CHIP and c-Myc depends on the N terminally located tetratricopeptide (TPR) domain of CHIP which has been implicated as a chaperone interacting motif. Moreover, inhibition of HSP90 chaperone activity by 17-AAG reduces c-Myc protein levels in a CHIP dependent manner. CHIP reduces the transcriptional activity of c-Myc proteins and decreases the transcript abundance of its target genes. Because levels of CHIP negatively correlate with those of Bcl2, Akt1 and β-catenin, and now in this report c-Myc, the present study may strengthen the view that CHIP is a tumour suppressor.

Keywords: c-Myc; CHIP (STUB1); 17-AAG; HSP70; HSP90.

Abbreviations: UPS, ubiquitin/proteasome system; CHIP, carboxyl terminus of HSC70 interacting protein; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; Ni-NTA, nickel- nitrilotriacetic acid; 17-AAG, 17-N-Allylamino-17-demethoxygeldanamycin; H & E, Haematoxylin and Eosin.


c-Myc is a transcription factor of the basic helix loop helix family (bHLH-LZ) and is a potent oncoprotein. By dimerizing with either Max or Miz-1 under different cellular contexts c-Myc can act as either a transcriptional activator or repressor respectively. The transcriptional targets of c-Myc includes genes involved in almost every aspect of cellular physiology ranging from cell cycle, cell growth, apoptosis, metabolism, immortalization to stem cells. It is expressed only in dividing cells and the mRNA and protein have short half lives (30 min and 20 min, respectively).

Over-expression of c-Myc is a hallmark of human cancers. It is over-represented in ~80% of breast cancers, and overall ~30% of all human cancers (Gardner et al., 2002). The over-abundance of c-Myc in cancer cells can be attributed to either transcriptional up-regulation or aberrant stabilization of the c-Myc protein or can be a combination of both. Genetic amplifications and/or translocations of the c-myc gene could increase c-myc transcript levels with a concomitant increase in c-Myc protein. Ectopic stabilization of the c-myc transcript is also an attractive possibility. However in certain cancers with c-Myc up-regulation no such aberration was detected. A large body of evidence has shown the definitive involvement of the ubiquitin/proteasome system in c-Myc degradation. Therefore it becomes necessary that the post-translational regulation of c-Myc be understood in the context of c-Myc turnover because the impairment of such pathways could lead to the high levels of c-Myc protein observed in various cancers (Meyer et al., 2008). Till date, six different E3 ligases have been implicated in regulating c-Myc protein stability through the ubiquitin-proteasome system (UPS): Skp2 (Kim et al., 2003; von der Lehr et al., 2003); Fbw7 (Welcker et al., 2004; Yada et al., 2004); TRUSS (Choi et al., 2010); SCFβTrCP (Popov et al., 2010); HectH9 (Adhikary et al., 2005) and Huwe1 (Zhao et al., 2008). Experimental loss-of-function of SCFSkp2, Fbw7, Huwe1 and TRUSS stabilized c-Myc whereas the functions of SCFβTrCP and HectH9 are required for c-Myc stability. TRUSS and SCFFbw7 decrease whereas SCFSkp2, SCFβTrCP and HectH9 increase c-Myc transactivation potential. Not all the E3 ligases reported are linearly regulated in cancer cells in terms of c-Myc activity. Furthermore the stabilizing mutations of c-Myc (Salghetti et al., 1999) observed in some cancers have not been documented in most others. Thus c-Myc turnover is complex and is critically determined by multiple mechanisms that are likely to differ under different cellular contexts.

Over-expressed c-Myc has been shown to co-localize with HSP70 in the nucleus of COS, HeLa, 293 and CV1 cells (Henriksson et al., 1992). However, the fate of c-Myc upon such interaction is not understood. Endogenous HSP90 was shown to physically interact with c-Myc and was found to be tethered with c-Myc transcriptional complex on the Connexin43 promoter in NIH3T3-Ras transformed cells. Treatment with geldanamycin reduced c-Myc driven transcription of Cx43 promoter-luciferase reporter (Carystinos et al., 2003). Interestingly, the possibility of a direct involvement of the chaperones on c-Myc protein stability has not been explored.

The carboxyl terminus of HSP70-interacting protein (CHIP) is a U box containing E3 ligase that links the chaperone and the 26S proteasome machinery by ubiquitinating chaperone substrates and marking them for degradation by the proteasome (Ballinger et al., 1999). Several independent reports have now established that the observed down-regulation of many HSP90 clients such as CFTR (Meacham et al., 2001; Okiyoneda et al., 2010), LRRK2 (Ding et al., 2009; Ko et al., 2009) and RUNX2 (Li et al., 2008) upon 17-AAG (a derivative of geldanamycin) treatment is in large part dependent on CHIP activity. These observations prompted us to ask whether c-Myc protein stability is affected as a result of its partnership with the chaperones and whether CHIP can act as an E3 ligase of c-Myc in this context. In this report, we show that CHIP induces the proteasomal-dependent degradation of c-Myc and thus identify a novel mechanism for c-Myc protein level regulation. We found that CHIP interacts with and ubiquitinates c-Myc leading to the latter's proteasomal degradation. In addition, we found CHIP to be a critical determinant of c-Myc half-life. Thus CHIP can be a potentially valid candidate for treating c-Myc driven cancers.


1. CHIP physically associates with c-Myc

To investigate the possibility that CHIP is involved in c-Myc ubiquitination and degradation, we first examined whether CHIP could interact with c-Myc. We transfected his-tagged c-Myc and FLAG-tagged CHIP alone or in combination and subjected to pull-down assays. As shown in Figure 1a, Ni-NTA beads could immunoprecipitate both exogenous and endogenous CHIP in HEK293T cells. CHIP was successfully co-precipitated with Ni-NTA beads in the presence of c-Myc-myc-his but not in its absence. As shown in Figure 1b, the interaction was substantiated by reciprocal co-immunoprecipitation using FLAG antibody. This suggested that CHIP and c-Myc can co-exist in the same complex. The structure of CHIP comprises three well characterized domains: (i) an N-terminal region consisting of three tandem TPR (tetratricopeptide repeats) motifs implicated in protein-protein interaction and are responsible for the interaction of CHIP with the molecular chaperones HSP70 and HSP90 (Ballinger et al., 1999), (ii) a central coiled-coil domain important for dimerization (Nikolay et al., 2004) and (iii) a C-terminal U box domain that harbors' the E3 ligase activity of CHIP (Hatakeyama et al., 2001). To determine the region of CHIP important for its interaction with c-Myc we transfected either the full length CHIP (CHIP-WT) or deletion mutants lacking either the TPR domain (CHIP-∆TPR) or the U box domain (CHIP-∆U box) in HEK293T cells together with c-Myc. Immunoprecipitation with anti-c-Myc antibody revealed efficient interaction between c-Myc and CHIP-WT and CHIP-ΔU box the interaction with CHIP-ΔTPR was completely lost suggesting that the TPR domain of CHIP was necessary for its interaction with c-Myc. If CHIP and c-Myc should interact they must co-localize inside the cells. To determine this we used double-labeling immunofluorescence microscopic analysis in DBTRG-05MG cells. We found that a portion of both c-Myc and CHIP co-localized in these cells (Fig. 1d). Collectively, these findings support a physiological interaction between CHIP and c-Myc.

2. c-Myc Is a Substrate for the CHIP E3 Ligase Activity

CHIP has been reported to ubiquitinate many chaperone clients targeting them for proteasomal degradation. Our observations above led us to speculate that c-Myc might be a substrate for the E3 ligase activity of CHIP. To determine this we undertook in vivo ubiquitination experiments in HEK293T cells transfected with c-Myc and ubiquitin together with either CHIP-WT, CHIP-∆TPR or CHIP-∆U box and performed immunoprecipitation with anti-c-Myc antibody. Immunoblotting with anti-ubiquitin antibody revealed substantial ubiquitination of c-Myc in case of CHIP-WT but not in case of CHIP-ΔTPR. However, we noted a strong ubiquitination of c-Myc upon CHIP-ΔU box over-expression (Fig. 2a). If CHIP mediated ubiquitination leads c-Myc towards proteasomal degradation then blockade of the proteasome will be expected to stabilize ubiquitin-conjugated c-Myc levels. Indeed treatment with MG132, a known proteasomal inhibitor, augmented immunoreactivity with ubiquitin antibody upon immunoprecipitation with anti-c-Myc in cells co-transfected with CHIP-WT as compared to cells transfected with empty vector (Fig. 2b). We also show that over-expressed CHIP efficiently incorporates poly-ubiquitin chains comprising Ub-WT but not Ub-KO which lacks all internal lysine residues (Fig. 2c). These observations indicate that CHIP poly-ubiquitinates c-Myc thus marking it for proteasomal degradation.

3. CHIP regulates steady-state levels of c-Myc protein

To explore whether CHIP can modulate the turnover rate of c-Myc, cells were co-transfected with c-Myc and either CHIP-WT or empty vector. 24 hours after transfection all cells were treated with 2.0 µg/ml cycloheximide (CHX), a broad-spectrum protein synthesis inhibitor, for indicated periods of time. Western blot analysis in Figure 4a shows that half-life of c-Myc proteins were attenuated in presence of CHIP. Conversely, if CHIP accelerates c-Myc degradation then down-regulation of CHIP should stabilize c-Myc. Consistent with this we found that CHIP siRNA treated cells retain endogenous c-Myc proteins better than cells treated with control siRNA (Fig. 4b). Thus knock-down of CHIP increases steady-state levels of endogenous c-Myc. Taken together these findings indicate that c-Myc stability is critically regulated by CHIP. To test the possibility that the observed down-regulation of c-Myc is at the transcriptional level we performed RT PCR analyses and found a modest decrease of c-Myc transcript levels upon CHIP over-expression, as shown in Fig. 3c. This indicates that CHIP can regulate the availability of c-Myc mostly at the protein level.

4. CHIP and HSP70/90 regulate c-Myc in an interdependent manner

Previous reports have hinted that inhibition of HSP90 by 17-AAG treatments decreases c-Myc protein. To determine this and whether CHIP is involved in the observed phenomenon we treated cells with an increasing concentration of 17-AAG, a potent HSP90 inhibitor, and monitored the levels of c-Myc by western blotting. As shown in Figure 4a, HSP90 and HSP70 levels increased upon 17-AAG treatment but c-Myc levels declined as expected. Next, to determine the role of CHIP we transfected 17-AAG treated cells with both scramble siRNA or CHIP siRNA and found that 17-AAG reduced c-Myc levels in cells transfected with scrambled siRNA, but siRNA suppression of CHIP abrogated the 17-AAG-mediated c-Myc reductions (Fig. 4b). Furthermore, the absence of an additive effect of 17-AAG and over-expressed CHIP in reducing c-Myc levels suggests that they function in the same pathway (Fig. 4c). Thus it seems that HSP90 is required for the stability of c-Myc and both 17-AAG and CHIP have the opposite effect of de-stabilizing c-Myc. To determine whether c-Myc associates with CHIP directly or instead by a chaperone intermediate we ectopically over-expressed either CHIP-WT or various mutants of CHIP (CHIP-ΔTPR and CHIP-K30A), compromised for their interaction with HSPs. Co-immunoprecipitation experiments using c-Myc as the bait protein (Fig. 4d) revealed that c-Myc could co-precipitate CHIP-WT efficiently but failed to do so with CHIP-∆TPR and very weakly with CHIP-K30A. We also found that HSP70 co-precipitated with c-Myc to a much greater extent than did HSP90 suggestive of a very transient nature of their partnership. As corroboration to these findings western blot in Figure 4e shows that transiently over-expressed CHIP decreased c-Myc in these cells whereas no such effect was observed in case of CHIP-K30A (which does not dock with chaperones) and CHIP-H260Q (U box mutant). Our observations showing the interaction between c-Myc and the chaperones demanded that they must have the opportunity to come in close association with each other in intact cells. To test this we performed double-labeling immunofluorescence microscopy in DBTRG-05MG cells and found that indeed c-Myc and both HSP70 and HSP90 co-localized. All these data indicate that CHIP and the chaperones play interdependent roles in c-Myc degradation and that both the chaperone binding and E3 ligase activity if CHIP is required for the observed phenomenon.

5. CHIP decreases c-Myc transcriptional activity

Because CHIP could decrease c-Myc protein levels we asked how it affects the transactivation potential of c-Myc. First, we measured mRNA levels of well known c-Myc target genes using real-time RT PCR analysis upon CHIP over-expression and found significant reductions in their relative expressions (Figure 5a). Next, to verify this effect we used a c-Myc responsive luciferase reporter vector and measured c-Myc transactivation (in terms of relative light units) in response to CHIP over-expression. As shown in Figure 7b-d, over-expression of c-Myc in HEK293T cells induced a ~3.5 fold transactivation whereas over-expression of CHIP alone reduced promoter activity below the basal level (suggestively by decreasing endogenous c-Myc proteins). Co-expression of CHIP-WT but not CHIP-ΔU box with c-Myc compromised c-Myc mediated transactivation potential. These results indicate that CHIP is a crucial regulator of c-Myc transcriptional activity.

6. CHIP is down-regulated in rat C6 glioma model

According to The Cancer Genome Atlas project a major proportion (74%) of glioma cases display elevated patterns for a core set of genes including EGFR, ErbB2, FOXO and Akt (Anon, 2008). Interestingly almost all of them have been shown to be targeted by CHIP for degradation (Lissanu Deribe et al., 2009; Xu et al., 2002; Li et al., 2009; Dickey et al., 2008). Since in this report we propose c-Myc to be a substrate of CHIP we were interested to examine the relative levels of CHIP in normal versus glioma tissues. For this we decided to use the rat C6 glioma cell line and Sprague-Dawley rats partly because of the easy availability of normal and tumour brain samples in enough quantities we required to perform various experiments. Tumour formed by C6 glioma cells exhibit closest morphological resemblance to human GBM and has the advantage over simplified models that inflammatory and vascular mechanisms are activated. The model has been extensively discussed elsewhere (Grobben et al., 2002). Sections were prepared from C6 tumour tissue such that they carry an adjacent portion of normal tissue as well to eliminate handling variations (Figure 8a; first column). To delineate normal from cancerous region we first stained the tissues using H & E stain to examine gross morphological features (Figure 8a; second column). In parallel, we examined the expression of PCNA and GFAP using immunohistochemical staining procedure. We found a more than ~2.3 fold increase in PCNA expression in tumour region as compared to the normal region. We could not detect any GFAP reactivity in the cancerous portion agreeing with previous reports (Whittle et al., 1998; Nagano et al., 1993) (Figure 8a; third and fourth column). We then examined the expression of CHIP. As per our hypothesis we found a marked decrease in CHIP levels (more than ~25 fold) in cancerous portion as compared to the normal region (Figure 8a; fifth column). We then asked the question whether the observed down-regulation of CHIP was at the protein or at the transcript level. For this we performed real time RT PCR analysis of RNA isolated from normal rat astrocytes (NRA) and C6 cells. We found a ~30% decrease in CHIP mRNA levels in C6 cells compared to NRA (Figure 8b). These findings were supported by western blot analysis (Figure 8c). All these data cumulatively suggests that CHIP is significantly down-regulated in rat C6 glioma model.


c-Myc protein regulation is redundant with multiple E3 ligases acting on c-Myc under different cellular contexts and with different outcomes in terms of its transactivation potential. This study identifies CHIP as a novel E3 ligase of c-Myc. Unlike the three previously reported E3 ligases (see introduction) implicated in c-Myc regulation CHIP is the only U box containing and chaperone associated protein. Using co-immunoprecipitation and double-labeling immunofluorescence microscopy we show that CHIP and c-Myc can co-exist in the same complex. The TPR domain of CHIP was necessary for this interaction. We show that c-Myc is a substrate for the E3 ligase activity of wild-type CHIP but not CHIP-∆TPR. Interestingly, CHIP lacking the U box domain (CHIP-ΔU box) was still able to increase the ubiquitinated form of c-Myc. Similar result was also observed previously (Li et al., 2008; Ko et al., 2009). A possible explanation would be the involvement of other factors in the ubiquitination process. However, we show that the CHIP-K30A and CHIP-H260Q mutants were unable to degrade c-Myc as efficiently as CHIP-WT. This suggests that the E3 ligase activity of CHIP is indeed required for the degradation of c-Myc whereby the function of other factors may be auxiliary. Over-expression of CHIP decreases steady-state levels of c-Myc by polyubiquitination and subsequent targeting to 26S proteasomes as indicated by an increased polyubiquitinated form of c-Myc in presence of MG132. We found that over-expression of CHIP accelerates the rate of c-Myc degradation whereas knockdown of CHIP using RNAi approach stabilizes endogenous c-Myc. Because CHIP is a chaperone associated ubiquitin ligase we wanted to assess the involvement of HSP70 and HSP90 in the mechanism. Treatment with gradually increasing concentrations of the geldanamycin derivative 17-AAG resulted in a dose dependent increase of the chaperones HSP70 and HSP90 as expected with a concomitant reduction of c-Myc levels. There was no effect on the levels of either CHIP or GAPDH. Our co-immunoprecipitation experiments using c-Myc as the bait revealed that the CHIP deletion mutant lacking the TPR domain does not interact while the interaction with CHIP-K30A mutant is seriously hampered. Interestingly we found a very weak interaction between c-Myc and HSP90 suggesting a very transient nature of their interaction. On the other hand the interaction between c-Myc and HSP70 was much stronger. This suggests that the HSP70-bound pool of CHIP may be more important for c-Myc degradation than HSP90-bound CHIP.

Our findings have particular implications for cancer. The molecular chaperones (MC) together with the UPS form the so-called Protein Quality Control (PQC) system of the cell. The PQC is responsible for maintaining the cellular protein homeostasis. Cancer cells are thought to bias this system towards a net increase in tendency for proliferation. Several observations support this. In normally dividing cells HSP70/90 contributes ~2% of all cellular proteins which is further elevated in tumors. Cancer cells are much more vulnerable to inhibition of this system than their normal counterparts. Quite naturally, the HSP90 inhibitor 17-AAG (along with 10 others) and the proteasome inhibitor Bortezomib (along with 2 others) are in phase III clinical trials. Because a major molecular mechanism of carcinogenesis is the over-expression of oncoproteins and numerous of them being HSP90 clients it is not illogical to assign an anti-tumorigenic role for CHIP. Indeed, a recent study has found negative correlation between CHIP levels and the malignancy of human breast tissues. In this report we found that CHIP levels were immunohistochemically low to undetectable in most clinical Glioma specimens tested. c-Myc

We first compared the correlation between CHIP mRNA levels and tumour grade using the Oncomine database, which is a publicly available cancer gene expression datasets repository. Nine of 21 datasets that contain profiles classified by Brain and CNS cancer, showed an inverse correlation between CHIP mRNA expression and tumour grade. Two datasets characterized by large population sizes showed a significant inverse correlation between CHIP expression levels and tumour grade (Desmedt_Breast and vantVeer_Breast, P = 0.00035 and P = 0.00093, respectively). Analysis of expression profile datasets using another public repository ArrayExpress ATLAS also indicated similar inverse relationship between CHIP and tumour grade.

Materials and methods

Cell Culture, transfection and drug treatments

Human Glioma (DBT-RG 05MG) and normal (HEK293/T) and Rat Glioma (C6) cell lines were obtained from American Type Culture Collection (ATCC). Cells were cultured in D-MEM high glucose media supplemented with 10% heat-inactivated Fetal Bovine Serum and 2000 units/l of penicillin and 2 mg/l streptomycin (Invitrogen). All cells were maintained at 370C in a humid incubator with 5% CO2. Transfections of different plasmid DNAs were performed using either Calcium Phosphate Protocol (for 293T cells) or Lipofectamine 2000 (Invitrogen) (for cancer cells) according to the manufacturer's instructions. The following drugs/inhibitors were used: 17-AAG, MG132 and Cycloheximide (Calbiochem, Darmstadt, Germany). Concentrations used are given with the figures.


Expression plasmids

Full-length human CHIP (NM_005861) and c-myc (NM_002467) (both p67 & p64 isoforms) were amplified by PCR from total HEK293 cDNA prepared in our lab. The full length CHIP and two CHIP deletion mutants comprising amino acids 1-189 (CHIP-ΔU box) and 135-303 (CHIP-ΔTPR) were sub-cloned into pIRES-hrGFP-1a expressing FLAG as a C-terminal tag and an independent bicistronic GFP for visualization in mammalian cell transfections. The full length c-myc was sub-cloned into pcDNA3.1-myc-his. Point mutations of CHIP (CHIP-K30A and CHIP-H260Q) and that of c-myc (c-myc-T58A and c-myc-A44V) were generated using the QuickChange XL Site-Directed Mutagenesis kit (Stratagene). pL..HA-Ubiquitin-WT and HA-Ubiquitin-KO constructs were obtained from Addgene (Plasmid # respectively). The c-myc responsive promoter construct pGL3-MR was prepared in our lab the details of which are available upon request.

RNAi constructs

To generate CHIP shRNA, the target sequence was selected using the Whitehead Institute siRNA designing tool ( Both sense and antisense oligonucleotides were synthesized by IDT based on Addgene's protocol (, annealed and inserted into pLKO.1 expression vector (Addgene plasmid # ) between AgeI and EcoRI sites. Control shRNA was constructed similarly. All sequences are given in the supplementary material.

Ni-NTA pull down, Immunoprecipitation and Western blot analyses

Expression vectors were transfected into HEK293T cells using Calcium phosphate method for 36 h before lysis. For pull down using Ni-NTA (Qiagen) cells were lysed on ice in Lysis Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole). Lysates were incubated with 10 µl (of 50% slurry) Ni-NTA beads at 40C for 1 h then washed 3 times with lysis buffer containing 50mM imidazole. Complexes were eluted with lysis buffer containing 250 mM imidazole. For co-immunoprecipitation experiments using antibodies, cells were lysed on ice using Co-IP buffer (50 mM HEPES pH 7.2, 250 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1.0 mM EDTA, 0.5 mM DTT, 10 mM PMSF and protease inhibitor cocktail). After pre-clearing with Protein A Sepharose beads (Amersham Biosciences), 800-1000 µg of total protein was subjected to immunoprecipitation. For western blotting, cells were lysed on ice using Tris lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, 10% Glycerol + protease inhibitors). Homogenates were incubated on ice for 30 mins and then centrifuged at 14, 000 rpm for 10 mins at 40C. The supernatant were collected and aliquoted into fresh tubes and stored in -800C. Cytoplasmic/Nuclear extracts were prepared using NEPER kit (Pierce) according to the manufacturer's instructions.

Typically, 25-50 µg of each sample were loaded on SDS-PAGE and transferred to PVDF membranes (Millipore, USA) using Semidry Electroblotter (BioRad). Primary antibodies were diluted appropriately with TBS-T + 2.5% w/v BSA and incubated overnight at 4°C with gentle agitation. Secondary antibodies were incubated for 1 h at room temperature in TBS-T + 2.5% w/v BSA diluted to 1:2000. The immunoreactive bands were visualised by chemiluminescence (Amersham Biosciences). GAPDH or β-actin served as loading control for whole cell lysate, α-tubulin for cytoplasmic extract and lamin b for nuclear extracts.

c-Myc (Rabbit), Myc-tag, FLAG, CHIP, HSP90, HSP70 primary antibodies and all HRP-tagged secondary antibodies were purchased from Cell Signalling Technology. c-Myc (Mouse), GAPDH, α-tubulin and laminB were purchased from Santacruz Biotechnology.

Real-time PCR

The total RNA sample was extracted using the TRIzol reagent (Invitrogen). The isolated RNAs were stored with Ribolock RNase Inhibitors (Fermentas, USA) at -800C until use. Quality of samples was determined by agarose gel electrophoresis (integrity) and by A260/280 measurements (>1.8, purity). Only high quality samples were accepted and used for conversion to cDNA using High Capacity Reverse Transcription Kit (Applied Biosystems, USA). This cDNA pool was subsequently used for QRT-PCR reaction using Power SYBR Green Master Mix (Applied Biosystems, USA) as a 10 X mix. The primers used were synthesized by IDT. Amplification was carried out for 45 cycles using the 7500 Fast Real Time PCR system (Applied Biosystems, USA). In all experiments 18S rRNA served as the internal control (normalization) and calibrator controls were chosen appropriately. All sequences are given in the supplementary material.

Luciferase assays

HEK293T cells transiently expressing renilla luciferase and firefly luciferase reporter plasmids were subjected to the treatments indicated. Luciferase activity of cell lysates was determined luminometrically using an luminometer (Promega ) by the dual luciferase assay system (Promega) as specified by the manufacturer. The measured values were analyzed with WinGlow Software (Promega). Quantification was based on at least three independent experiments.


Immunofluorescence microscopy

For immunofluorescence microscopy cells were grown on cover slips placed inside 35 mm dishes, washed with PBS (+2mM MgCl2), fixed with 3.7% paraformaldehyde, quenched with 100 mM glycine and again washed with PBS. Permeabilization was done with 0.5% Triton-X100 and blocked with 3% BSA. Primary antibodies (1:200) against c-myc (Santacruz Biotechnology), CHIP, HSP70 and HSP90 (Cell Signalling Technology) were incubated overnight at 40C. FITC or TRITC tagged secondary antibodies (1:1000) (Molecular Probes) were incubated for 2 hours at room temperature. Cover slips were mounted using DAPI containing mounting media and sealed with nail polish. Photographs were taken on Olympus BX61 motorized fluorescence microscope using Image ProPlus software (Olympus).

Tumour samples and Immunohistochemistry

The specimens from human Glioma patients were provided by the Department of ……, Park Clinic, Kolkata, India. All tissues and/or sections were obtained following the regulations approved by the …………. The pathological diagnoses of all enrolled patients were confirmed by two different pathologists.

For immunohistochemistry formalin-fixed paraffin-embedded 3µm sections of human Glioma were obtained from the Park Clinic (Kolkata, India). Sections were processed following standard methods. Briefly, sections were deparaffinised in xylene and rehydrated in graded ethanol series. For antigen retrieval sections were dipped into citrate buffer (pH ) and pressure heated for 15 mins. After quenching endogenous peroxidise activity by 3% H2O2, sections were blocked with 3% BSA for 2 hours before incubation with primary antibodies against c-myc (Santacruz, 1:50), CHIP (Cell Signalling, 1:100) overnight at 40C followed by incubation with HRP tagged secondary antibody (Cell Signalling) at 1:1000 dilution for 1 hour. Sections were then stained with DAB (BD Biosciences) and counterstained with haematoxylin. To ensure specificity sections were kept where normal IgG antibodies replaced primary antibodies and served as negative controls. For evaluating the results

FACS experiments


We thank