2.1. Cell culture and treatment
The human hepatoma HepG2, the human pancreatic carcinoma PANC-1, and the human astrocytoma 1321N1 cells (American Type Culture Collection, Manassas, VA) were cultured in Minimum Eagle's medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin and 50 mg/ml streptomycin. Cells were serum starved for 4 h in MEM:F12 (1:1) followed by the addition of vehicle, PDTC (50 μM) or a potent Hsp90 inhibitor for the indicated times. The Hsp90 inhibitors used include novobiocin and the geldanamycin analogs, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and DMAG, a water-soluble analog of 17-AAG. PDTC, novobiocin, 17-AAG and DMAG were purchased from EMD-Calbiochem (San Diego, CA).
2.2 Protein crosslinking in intact cells with ethylene glycol bis(succinimidyl succinate) (EGS)
Cells were washed twice with phosphate-buffered saline and incubated with 1 mM EGS (Thermo Scientific Pierce, Rockford, IL) for 30 min at 25ºC, followed by quenching of the cross-linking reaction with the addition of 10 mM glycine. Cells were lysed in SDS sample buffer supplemented with 7.5% 2-mercaptoethanol, and the samples were processed for Western blot analysis.
2.3. Western blot analysis
Unless otherwise indicated, cells were lysed in RIPA buffer (He et al., 2006
) and proteins were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and then transferred onto polyvinylidene difluoride membranes. Western blots were performed according to standard methods, which involved the visualization of immunoreactive bands by enhanced chemiluminescence, their quantitation by volume densitomety using ImageQuant software (Molecular Dynamics, Piscataway, NJ), and normalization to GAPDH. In this study, the primary antibodies were directed against HSF1 (StressGen Biotechnologies, Victoria, Canada), BAG3 (Abcam, Cambridge, MA), Hsp90α (BD Biosciences, San Jose, CA), Hsp72, BRG1, p65Rel, IκBα, Mcl-1, and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) and were used generally at a dilution of 1:1000.
2.4. Preparation of nuclear extracts
Nuclear extracts from HepG2 cells were prepared using the NE-PER extraction kit (Thermo Scientific Pierce) and quantified using the bicinchoninic acid protein assay reagent.
2.5. RNA interference
Transfection of 21-nucleotide siRNA duplexes (Qiagen) for targeting endogenous HSF1 was carried out using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA) and 20 nM small interfering RNA (siRNA) duplex per 35-mm plate according to the supplier s instructions. Transfected HepG2 cells were assayed 3 days after reverse transfection. The sequences of HSF1 siRNA used were: r(GGUUGUUCAUAGUCAGAAU)dTdT (sense) and r(AUUCUGACUAUGAACAACC)dTdG (anti-sense), whereas the negative control siRNA was the “AllStars Neg. Control siRNA” (Qiagen) that has no known target gene. Specific target gene silencing was confirmed by real-time PCR and immunoblotting.
2.6. Reverse transcription and real-time PCR analysis
Total RNA was isolated and first strand cDNA was synthesized using the Omniscript Reverse Transcript kit (Qiagen). Real-time PCR reactions were carried out with the TaqMan® Gene Expression Assay system method on an ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA). Primer pairs used for the reactions are listed in . Relative quantitation of gene expression was performed using the threshold cycle. The mRNA levels were compared to standard curves (generated using serial dilutions of human HepG2 RNA) and differences in mRNA expression were calculated by the ΔΔCT method after normalizing to GAPDH mRNA.
List of primer identification number (ID) and size of the PCR products for real time PCR
2.7. Chromatin immunoprecipitation (ChIP) assay
HepG2 cells were treated with vehicle, PDTC (50 μM) or DMAG (5 μM) for 1 h, and ChIP assays were then performed essentially as described (Wurster and Pazin, 2008
). In brief, cells were crosslinked with 1% formaldehyde at room temperature for 10 min. After harvesting cells with SDS lysis buffer, DNA was sheared to 200–1000 bp by sonication, and the cell lysates were precleared by centrifugation. Polyclonal antibody developed against HSF1 (StressGen) was used to recover HSF1-bound DNA complexes. In addition, precleared lysates were also incubated with rabbit IgG as a control for potential non-specific coprecipitations. DNA was quantified by real-time PCR using promoter-specific primers for select genes (). Each experiment was repeated 3-5 times.
Promoter-specific primer sequences used for ChIP assays
2.8. Labeling of protein reactive thiols in cellular extracts
Cells were lysed in RIPA buffer supplemented with 200 μM maleimidobutyrylbiocytin (MBB, EMD-Calbiochem), an irreversible thiol-specific biotinylating agent, for 30 min on ice followed by the addition of an excess of L-cysteine (5 mM) to quench the reaction. An aliquot of the clarified lysates was subjected to Hsp90 immunoprecipitation and Western blotting. Polyvinylidene difluoride membranes were blocked with 1% polyvinylpyrrolidone (Sigma-Aldrich, St-Louis, MO) in TBS-T and incubated with horseradish peroxidase-conjugated streptavidin (Vector Laboratories, Burlingame, CA) for the detection of biotin-labeled protein thiols. A second aliquot was incubated with captavin-agarose beads (Invitrogen) for 1 h at 4°C to immobilize thiol-biotinylated proteins, which were then eluted with 50 mM NaHCO3 (pH 10.0). After neutralization, Hsp90 immunoprecipitation and Western blot analysis were carried out.
2.9. Statistical analysis
All results are expressed as relative to the control value. Experiments were performed in at least two to three different culture preparations, and two dishes for each experimental condition were plated in each preparation. Results are expressed as means ± SD, where indicated, with n reflecting the number of observations. Statistical comparisons between groups were made by unpaired Student s t-test. Analyses were performed using the software package Kaleidagraph v4.01 (Synergy Software, Reading, PA) with values ≤ 0.05 considered significant.