Control antibody

5-μm thick formalin-fixed and paraffin-embedded samples were processed for immunohistochemistry stainning with antibodies against human CD3 (1:500 dilution, Dako A/S, Glostrup, Copenhagen, Denmark) or control antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The density of CD3+ were evaluated quantitatively by the mean number at five representative fields at 400× magnification by two independent observers who were blinded to the clinical outcome. A significant linear correlation existed between the counts of two independent observers and the average counts of the two investigators was used in subsequent analyses to minimize inter-observer variability.

The assay was performed according to the manufacturer's protocol (ChIP-IT protein G magnetic beads kit, Active Motif, Carlsbad, CA). In brief, cells were seeded onto a 10 cm culture dish and one day later were cross-linked by 1% formaldehyde for 10 minutes at room temperature and followed by fragmentation of genomic DNA using a sonification apparatus. Cell lysates were used for chromatin immunoprecipitation using Bach1 (sc-14700, Santa Cruz, CA), HDAC1 (sc-7872, Santa Cruz, CA), Ac-Histone H3 (sc-56616, Santa Cruz, CA), and Control antibody (sc-2028, Santa Cruz, CA). The protein G magnetic beads-antibody/chromatin complexes were washed and the antibody/chromatin complexes were subsequently eluted. The cross-linked protein/DNA complexes were detached at 65°C for four hours followed by purification of the genomic DNA. PCR primers amplifying human VEGF promoters are shown in Table 1. Real-time PCR was carried out using SYBR Green PCR master mix. The amount of DNA associated with each protein (relative to the total amount of DNA used) was determined.

EZ-ChIP™ Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was purchased to perform the ChIP assay, according to the manufacturer's protocol. Briefly, one 15 cm cell culture plate of SW480 cells (about 80% confluence) was fixed with 1% PFA (Sigma). Then cells were lysed, sonicated to shear DNA and immunoprecipitated with anti-E2A (Santa Cruz) and control antibody (IgG). After that, protein/DNA crosslink was reversed and DNA was purified for the following PCR, using Takara Ex Taq Hot Start Version (Takara).

ChIP assays were performed with 2.5μg anti-Nrf2 (Santa Cruz, Dallas, Texas), anti-BRD3 (Bethyl Lab, Montgomery, Texas), anti-BRD4 (Bethyl Lab) or control antibody (Santa Cruz), followed by real-time PCR with primers targeting a negative control region or the promoter region of the HMOX1 gene [44 (link)]. Fold enrichment of the antibody at the HMOX1 gene promoter region was calculated by dividing PCR threshold from the anti-Nrf2, BRD3 or BRD4 antibody by PCR threshold from the control IgG, relative to the negative control region.

B(a)P, EN, ICI 118.551, salbutamol, propranolol and actinomycin D were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). Rabbit monoclonal antibody anti-β2ADR and control antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other compounds were commercial products of the highest purity available. Chemicals were used as stock solutions in DMSO; the final concentration of this solvent in culture medium was always <0.2% (v/v), and control cultures received the same dose of vehicle as exposed cultures.