Healthy adult volunteers were recruited to the NIEHS Clinical Research Unit and underwent phlebotomy. Subjects were excluded if they had a history of recent infection, were on anti-inflammatory medications, or tested positive for hepatitis B, C or HIV. Up to 300 ml of whole blood were withdrawn from an antecubital vein into citrated tubes. Lymphocytes were isolated using percoll (Sigma) and anti-CD3-coupled Magnetic Beads (Miltenyi Biotec) as per manufacturer's protocol. Cell purity was >98% after percoll/magnetic bead isolation based on flow cytometry. We maintained lymphocytes in RPMI supplemented with 10% FBS. For T cell stimulation, cells were activated with phytohemagglutinin-M (PHA, Invitrogen, 3% vol/vol) for 72 h. The total number of lymphocytes available per treatment conditions after PHA was typically around 10 million cells or less. Cells were treated starting at 48 h post PHA addition and cell cultures were harvested 24 h later. Freshly isolated CD3+ cells were treated with nutlin for 20 h or DMSO as a vehicle control, then washed and exposed to TLR1/2 ligand PAM3CSK4 (1 µg/ml) at 1×106 cells/ml. Total yield of CD3+ cells from a single subject was typically around 10–15 million cells or less. Protocol and procedures were approved by the NIEHS Institutional Review Board.
Alveolar macrophage isolation
Healthy, nonsmoking male volunteers, 18 to 40 yr of age, underwent fiberoptic bronchoscopy with lavage to procure alveolar macrophages. The screening procedures for each subject included a medical history, physical examination, and routine hematologic and biochemical tests. None of the subjects had a history of asthma, allergic rhinitis, chronic respiratory disease, or cardiac disease. Subjects were excluded from the study if they had suffered a recent acute respiratory illness and were asked to avoid exposure to air pollutants such as tobacco smoke and paint fumes. A fiberoptic bronchoscope was wedged into a segmental bronchus of the lingula. Six 50-ml aliquots of sterile saline were instilled and immediately aspirated. The procedure was repeated on the right middle lobe, again using 300 ml of saline. Samples were put on ice immediately after aspiration and centrifuged at 300 x g for 10 min at 4–8°C. Cells from all aliquots were pooled, washed twice with RPMI 1640, and re-suspended in RPMI 1640 at 1.0×106/mL. Total yield was typically around 10–15 million cells or less. Around 2.0×106 cells per well were seeded in a 12 well plate. After 2 hr, cells were washed twice with warm PBS and 2 ml of growing media was added to each well. Cells were then treated with nutlin or doxorubicin. Cells were harvested 24 hr post-treatment. The protocol and consent form were approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects. Prior to participation in the study, subjects were informed of the procedures and potential risks and each signed a statement of informed consent.
Cancer cell cultures
H1299 lung cancer cells (American Type Culture Collection) were routinely maintained following standard conditions and procedures for culturing mammalian cells. All cultures were incubated at 37°C with 5% CO2. p53 tetracycline inducible SaOS2 TET-off cell lines expressing the wild-type or the G279E mutant protein were cultured as described previously 
. p53 expression was kept “off” by 2 mg/ml doxycycline (Clontech). To induce the p53 expression, cells were washed 3X with phosphate-buffered saline (PBS) and placed in medium lacking doxycyline during 24 h.
Reagents and treatment conditions
For drug treatment and p53 activation, cells were incubated with doxorubicin (Sigma 0.3 µg/mL), nutlin3 (Sigma, 10 µM) and 5-fluorouracil (Sigma, 300 µM). For ionizing radiation treatment, cells were irradiated at 1.56 Gy/min from a Shepherd cesium irradiator in PBS at room temperature at final dose of 4 Gy. Where indicated cells were also pretreated 2 h with p53 inhibitor pifithrin-alpha (Sigma, 40 µM).
RNA isolation and gene expression analysis
Total RNA was isolated by RNEasy kit (Qiagen). Real-time PCR was performed in triplicate with Taqman PCR Mix (Applied Biosystems) in the 7000 ABI sequence Detection System (Applied Biosystems). All human and mouse primers were purchased from Applied Biosystems (information available upon request). For PHA stimulated lymphocytes and alveolar macrophages expression of TLR genes was normalized to 18S ribosomal RNA gene while for freshly isolated CD3+ T cells, the glucuronidase-beta gene was used for normalization.
Luciferase reporter assays
Pairs of complimentary oligonucleotides for the desired p53RE from selected TLR
s and containing restriction sites were cloned into the open reading frame of firefly luciferase pGL4.26 plasmid (Promega) previously double digested by Xho I/Kpn I restriction enzymes. The identity of the inserts was confirmed by DNA sequencing. Luciferase activity was measured 48 h after Fugene6- mediated co-transfection of the TLR
p53RE constructs in the presence of p53 (pC53-SN3) or empty vector pCMV NEO-BAM3 along with pRL-TK Renilla as a transfection efficiency control into p53 null H1299 cells, as previously described 
. Forty-eight hours post-transfection extracts were prepared using the Dual Luciferase Assay System (Promega) following the manufacturer's protocol and luciferase activity was measured on a Victor Wallac multilabel plate reader (PerkinElmer). Relative luciferases activities for each construct was defined as the mean value of the firefly luciferase/Renilla luciferase rations obtained from 4 independent experiments performed in triplicate.
Whole cell extracts were quantified using the Bradford protein assay kit and gamma globulin as a reference standard (BioRad). For TLR protein detection, cellular pellets were subjected to subcellular protein fractionation (Thermo Scientific) following the manufacturer's instructions and protein was quantified using BCA protein assay kit (Thermo Scientific). For TLR western blot analysis ~30 µg of total membrane fraction was used, while for the analysis of other proteins ~25 µg of total cell extract were used. As expected, we did not detect TLR2 and TLR5 in the cytosolic fractions; therefore, those data are not included. Proteins were resolved on 4–12% BisTris NuPAGE and transferred to polyvinylidene difluoride membranes (Invitrogen) and were visualized with primary antibodies followed by horseradish peroxidase–conjugated goat anti–mouse or donkey anti-goat immunoglobulin (Santa Cruz Biotechnology) through the use of enhanced chemiluminescence reagents (Amersham Biotechnology). The primary antibodies used in these studies were against p53 (DO1, Santa Cruz Biotechnology,), p21 (SXM30, BD Biosciences Pharmigen) and Actin (C-11 Santa Cruz Biotechnology). The following is the list of TLR antibodies tested in this study in order to detect TLR protein expression in whole cell extracts as well as membrane and cytosol protein fractions: TLR8 ab24185 and TLR10 ab45088 from Abcam, Inc.; TLR1#2209, TLR2#2229, TLR7#2633 and TLR9#2254 from Cell Signaling. We also used a Toll-like receptor detection kit that includes antibodies for all human TLRs (TLR1 to TLR10 antibodies from ProSci, Inc., as well as TLR3-4H270 and TLR5-H1-27 antibodies from Santa Cruz Bitoechnology. The TLR3-IMG-315A, TLR4-IMG6370A antibodies were from IMGENEX. Only the TLR2 (Cell Signalling) and TLR5 (Santa Cruz) gave clear results. Attempts to detect other TLRs with these antibodies were unsuccessful and appear to be a general problem with TLR antibodies from our collective experience. One of the antibodies enabled us to detect induction of full length TLR4 by nutlin; however, those results are not presented due to the appearance in both untreated and treated samples of nonspecific bands.
Chromatin Immunoprecipitation (ChIP) assays
ChIP assays were done as previously described 
using ChIP kits (Millipore). Approximately 40×106
PHA stimulated lymphocytes were used for each experimental sample. Cell lysates were sonicated using conditions that yield chromatin fragments 200–500 bp long. One microgram of DO-7 p53-specific monoclonal antibody (BD Biosciences Pharmigen) was used per ChIP assay. As a negative control, mouse Ig (Santa Cruz Biotechnology) was used. PCR amplifications were performed on immunoprecipitated chromatin using primers to amplify specific regions on the TLR
s promoters (sequence information available upon request). The PCR cycles were as follows: initial 10 min Taq polymerase (Invitrogen) at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The PCR products were then run on a 1.8% agarose gel.
Flow cytometry analysis
Cells were resuspended in 100 µl of PBS and incubated with 5 µl of fluorescent antibody per sample for 30 min, then washed and fixed with 0.5% paraformaldehyde. The fluorescence intensity was evaluated using a Becton Dickinson LSR II Flow Cytometer. All antibodies used for FACS were from BD Pharmigen.
SNPs were assessed by three different approaches. In RFLP assays, genomic DNA was extracted from Percoll-isolated lymphocytes by DNeasy kit (Qiagen). For the SNP in the TLR8 p53RE#6 (AGGCAAGATGAAACAT(G/C)TCA), the G-SNP creates a unique restriction cutting site for NspI (R CATG Y). PCR was performed with 100 ng of DNA, 50 pmol of each primer, 1.5 mM MgCl2, 1 µL 10× PCR buffer, and 0.0125 U of Taq (Invitrogen). After 10 min at 94°C, 35 cycles were repeated as follows: 94°C 30 s, 60°C 30 s, and 72°C 35 s; this cycling was followed by a final extension at 72°C for 10 min. The PCR product was digested with 5 U Nsp I (New England Biolabs, Ipswich, MA), at 37°C for 4 h. Since Nsp I recognizes the polymorphic sequence, a G allele is demonstrated by the presence of two fragments 109 and 69 bp in a gel. The A allele is revealed by the presence of a single 177 bp band. The following primers were used for amplifying the region containing the p53RE on TLR8 promoter region:
The status of this SNP was determined also by using a Taqman SNP genotyping assay. All primers were purchased from Applied Biosystems (Assay ID:C_27497635_10). For direct sequencing the region containing the p53RE was first PCR amplified using the following pairs of primers:
This was followed by running the samples on a TEA-agarose gel. The expected product (397 bp) was cut out and cleaned using QIAquick gel extraction kit (QIAGEN). The sequencing reactions used Big dye (Applied Biosystems) per manufacturer recommendations and the following primers:
Mouse primary cell culture
p53+/+ and p53−/− mouse embryonic fibroblasts (MEFs) were cultured in DMEM media and 10% of FBS. Female C57BL/6 mice were purchased from Jackson Laboratories. All experiments were performed in accordance with the Animal Welfare Act and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals after review of the protocol by the Animal Care and Use Committee of the National Institute of Environmental Health Sciences. For murine peritoneal macrophage harvests, mice were injected i.p. with 2 ml of 4% Brewer's thioglycollate and euthanized 96 h later. The peritoneum was washed with 10 ml ice cold PBS three times. Cells were centrifuged (1,000x RPM, 6 minutes, 4°C) and washed twice with sterile PBS. Peritoneal exudate macrophages were resuspended in DMEM/0.1% FBS, counted, and plated at 2×106 cells/well in a 12-well plate. Cells were allowed to settle for 2 h (37°C/5% CO2) before replacing media with DMEM complimented with 10% FBS.
For bone marrow-derived macrophages (BMM), marrow was flushed from femoral and tibial bones using bone marrow media (DMEM/2 mM L-glutamine/10% L929-conditioned medium/10% FBS). Cells were spun down (2200x RPM, 5 min, 4°C), brought up in 1 ml sterile ACK buffer, incubated 4°C for 1 min after which 10 ml PBS was added. Cells were spun as above, resuspended in bone marrow medium, counted, and plated at 1×106 cells/well in a 12-well plate. Cells were cultured at 37°C and 10% CO2 in 2 ml bone marrow medium/well and fed on Day 5 with addition of 1 ml medium/well. Experiments were performed on Day 6. At 24 h post-treatment, cells were harvested for RNA extraction.
To examine statistically whether the average mRNA fold-change at each locus in the population sampled differed from 1 for the various exposures, we applied one-sample Student's t tests to log-transformed values of mRNA fold change. The logarithmic transformation helps the data meet the distributional assumptions for the t test. This procedure, in effect, tests the null hypothesis that the geometric mean mRNA fold change at the locus is equal to 1 against the two-sided alternative that the geometric mean differs from 1.