Cell culture and treatment
The mouse melanoma B16F10 cells were grown in RPMI (Sigma) medium supplemented with 10% fetal bovine serum (FBS). Heat shock was performed at 42,5°C. For the EMSA experiment duration of heat shock was 30 min and 1 h, without recovery at 37°C. For microarray analysis duration of heat shock was 1 h at 42,5°C with 30 min recovery at 37°C. LA treatment was done as described in "Transfections" but without adding plasmid DNA. For the EMSA experiment cells were treated with Lipofectin (Gibco) or Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco BRL) medium for 1 h and 2 h and processed immediately. For microarray analysis of LA effect, cells were treated with Lipofectamine 2000 (Invitrogen) for 3 h in Opti-MEM (Gibco BRL) medium and then the liposomes containing medium was replaced with RPMI supplemented with 10% FBS and cells were allowed to recover for 30 min at 37°C.
The transient transfections have been performed using the following compounds: DEAE-Dextran (Pharmacia), Lipofectin (1:1 DOTMA and DOPE, Gibco BRL), Lipofectamine 2000 (3:1 DOSPA and DOPE, Invitrogen) and two liposome formulations prepared in our Institute DDAB/DOPE  and Arg-Chol/DOPE .
For transient transfection using DEAE-Dextran, cells were seeded at 3 × 105 cells on a 90 mm plate. The next day, cells were treated with the mixture containing 10 μg plasmid DNA and 1 mg/ml DEAE-Dextran in TBSP buffer (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.5 mM MgCl2 and 1 mM Na2HPO4) at room temperature for 30 min. Then cells were washed with a TBSP buffer and treated with RPMI with 10% DMSO for 2 min. Cells were washed with DMSO-free TBSP buffer, then medium supplemented with FBS was added and cells were incubated at 37°C in a CO2 incubator for 6-48 h.
Transient transfections using liposome formulation DDAB/DOPE, Arg-Chol/DOPE and Lipofectin were done according to standard procedure. Cells were seeded at 2 × 105 cells on 60 mm plate a day before. For each transfection, 10 μg plasmid DNA was diluted in 100 μl Opti-MEM medium (Gibco BRL) and added to liposome dilution. Liposome dilution was prepared by adding 20 μl of liposomes (1 mg/ml) into 100 μl Opti-MEM and followed by incubation at room temperature (RT) for 45 min. DNA-liposome mixture was incubated for 15 min at RT. While complexes were forming, the cells were washed twice with serum-free Opti-MEM medium and then overlaid with that medium. DNA-liposome complexes were dropped onto the plate. Cells were incubated at 37°C in a CO2 incubator for 3 h. Then the medium was changed for RPMI supplemented with FBS and cells were incubated at 37°C in a CO2 incubator. Cells were either grown for 6 or 48 h, or after 24 h at 37°C heat shocked for 45 min at 42,5°C followed by recovery for 24 h. For kinetics study, incubation times were 6 - 48 h.
Transient transfection using Lipofectamine 2000 was done according to the supplier's instruction. Cells were seeded at 1,2 × 105 cells on a 30 mm plate a day before. For each transfection, 4 μg of plasmid DNA was diluted in 250 μl of Opti-MEM and added to liposome dilution. Liposome dilution was prepared by adding 10 μl of Lipofectamine 2000 into 250 μl Opti-MEM and following incubation at RT for 5 min. DNA-liposome mixture was incubated for 30 min to allow formation of DNA-liposome complexes. The next steps were done as described above for transient transfection using Lipofectin and other liposome formulation.
Constructs used for functional analysis of the rat Hspa1b promoter contained 85 bp of the 5'UTR and different fragments of the promoter region, cloned in pBL-CAT6 vector bearing the bacterial chloramphenicol acetyltransferase (CAT) reporter gene (Figure 4) . pR70/GFP plasmid contained PstI-AvaI restriction fragment (from nt -870 to nt +85) of the rat Hspa1b promoter cloned into the SalI and BglII sites of pEGFP-1 Promoter Reporter Vector (Clontech) .
Electrophoretic mobility shift assay
Nuclear extracts were prepared as described by Suzuki et al. . Nuclei isolated from cells were lysed by Nonidet-P40 and proteins were extracted with buffer consisting of 0.35 M NaCl, 5 mM EDTA, 1 mM DTT, 10 mM Hepes, pH 7.9 and 0.2 mM PMSF by incubation on ice for 20 min. DNA-protein binding activity was analyzed by incubating nuclear extracts with [α-32P]-labeled double stranded HSE oligonucleotide (HSE consensus sequence):
Binding buffer contained 20 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 1 mM EDTA, 0.2 M NaCl, 2 μg poly(dI-dC). The HSE-protein complexes were separated on a native 6% polyacrylamide gel. Gels were dried and exposed to an X-ray film (Kodak).
Cell culture extracts were prepared by the Tris buffer freeze-thaw protocol. Protein concentration was determined by Bradford method. The CAT activity was detected as described in . Briefly, 25 μg of protein from cell extracts were incubated with 14C-labeled chloramphenicol (ICN) and acetyl-coenzyme A (Sigma) for 4 h at 37°C. The reaction products were separated by thin layer chromatography, then plates were exposed to X-ray film (Kodak). For quantitative analysis, CAT activity was monitored by scintillation counting; counts were converted to the percentage of acetylated chloramphenicol as described in . Data for each experimental point was gathered in quadruplicate.
Total RNA was isolated using RNeasy Mini Kit (Qiagen) with on-column DNA digestion using DNAse I (Qiagen) according to the manufacturer's recommendations. RNA quantity was estimated with ND-1000 spectrophotometer (NanoDrop Technologies). RNA quality was assessed using Agilent platform: RNA 6000 Nano LabChip Kit, RNA Integrity Number software and the Agilent 2100 Bioanalyzer (Agilent Technologies).
We used the Mouse Expression Arrays 430A (Affymetrix). The hybridization target was prepared according to the recommendations from microarrays' manufacturer. Total RNA (8 μg) was used for synthesis of double stranded cDNA. Half of the cDNA volume was used for synthesis of biotinylated cRNA with the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). Both cDNA and cRNA were purified with Gene Chip Sample Cleanup Module (Affymetrix). cRNA (16 μg) was fragmented and hybridized to the microarray for 16 h at 45°C. After washing and staining, the microarrays were scanned with GeneChip Scanner 3000 (Affymetrix). Data was acquired using GCOS 1.2 software (Affymetrix). The preprocessing was performed by Robust Multiarray Analysis (RMA, Bioconductor). Microarray data are available via NCBI GEO: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE29743.
Supervised gene selection was carried out by BRB Array Tools (developed by R. Simon and BRB Tools Development Team). Genes differentiating three analyzed groups were selected using a random-variance F-test. A stringent significance threshold was used to limit the number of false positive findings: differences in the expression of genes were considered statistically significant if their p value was less than 0.001. We also performed a global test of whether the expression profiles differed between the classes by permuting the labels of which arrays corresponded to which classes. For each permutation, the p values were re-computed and the number of genes significant at the 0.001 level was noted. The proportion of the permutations that gave at least as many significant genes as with the actual data was the significance level of the global test. False Discovery Rate (FDR) was estimated by Benjamini-Hochberg algorithm. Gene group analysis was carried out to obtain p values reflecting the differential expression of gene groups among classes. Four different tests: the Fisher (LS) statistic, Kolmogorov-Smirnov (KS) test, GSA test and Goeman test were used, as implemented in BRB Array Tools. We considered a GO category significantly differentially regulated if either significance level was less than 0.005, all categories with between 5 and 100 genes represented on the array were considered; some of the groups were overlapping. Promoter analysis was carried out using the Molecular Signatures Database, MSigDB. The MSigDB Predicted Promoter Motifs ontology contains sets of genes that share a transcription factor binding site in their promoters. The binding sites are predicted based on definitions from the TransFac database.