Age matched AKR and DBA/2 ApoE−/− mice were maintained on chow diet and sacrificed at 16 weeks of age. Plasma total cholesterol levels at time of sacrifice were 372.8±47.78 mg/ml and 912.7±104.3 mg/ml for AKR ApoE−/− and DBA/2 ApoE−/− mice, respectively. Femurs were collected to isolate and culture bone marrow monocytes.
All experiments were performed in accordance with protocols approved by the Cleveland Clinic Institutional Animal Care and Use Committee.
Total, free, and esterified cholesterol quantification
Bone marrow derived macrophages (BMM) from AKR and DBA/2 ApoE−/−
mice were cultured in p100 cell culture dishes for 13 days with 15% L-cell conditioned media, a source of macrophage colony stimulating factor (M-CSF). We performed control studies to compare BMM cholesterol loading by 24 h incubation with 50 µg/ml AcLDL, copper oxidized LDL, or native LDL; and, we only observed robust cholesterol loading with AcLDL at this dose, justifying its use in our subsequent studies. The cells were loaded for 48 h with 50 µg/ml AcLDL to allow foam cell formation with unloaded cells used as controls. Lipids were extracted and total, free and esterified cholesterol levels were quantified and normalized to cell protein as previously described 
. Quadruplicate samples were assayed in triplicate and significance was determined by ANOVA with Newman-Keuls posttest using GraphPad Prism software.
Loading of macrophages with acetylated LDL for transcriptome profiling
Differentiated BMM in p100 dishes from AKR and DBA/2 ApoE−/− mice were incubated for 24 hours in the presence or absence of 50 (experiment 2) or 100 µg/ml (experiment 1) AcLDL in quadruplicate. Different AcLDL doses were used due to altered efficiency of cholesterol loading of independent AcLDL preparations. After the incubation, the media and cholesterol were aspirated off, cells were washed twice, scrapped in 1 ml ice-cold PBS and transferred to microcentrifuge tubes. Cell pellets were obtained by centrifugation in a microcentrifuge at 5,000 rpm for 2 minutes at RT and kept on ice for subsequent use.
Isolation of total RNA from BMM cell pellets
Total RNA was isolated from cell pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA), following the manufacturer's protocol. On-column digestion with RNase-free DNase (Qiagen, Valencia, CA) was performed to remove genomic DNA. DNase was removed in the subsequent washing steps. RNA integrity was tested by overnight incubation of 200–500 ng of total RNA at 37°C and observation of the 18S and 28S ribosomal RNA bands on a 1.2% agarose/ethidium bromide gel.
Hybridization and detection of gene transcripts
An aliquot of total RNA for each sample (~ 2 µg) was submitted to the Genomic Core at Lerner Research Institute. Total RNA was used as template to synthesize single stranded cDNA following the Illumina protocol. Single stranded cDNA is then converted into double stranded cDNA, purified and in vitro transcribed (in the presence of biotinylated UTP and CTP) to produce biotin-labeled cRNA. Purified labeled cRNA from experiment 1 and 2 samples were hybridized respectively to MouseRef–8_v1 and Ref-8_v2 microarray chips (Illumina) for 16 hours at 58°C, according to the manufacturer's instructions. Eight samples were profiled on one chip with either 24,613 or 25,697 transcript probes per sample. To reduce the chip-to-chip variability two control and two cholesterol-loaded samples from each strain were put on each microarray chip. After hybridization, the arrays were washed several times and stained with streptavidin-Cy3. After the staining, the arrays were washed and scanned on the Illumina BeadArray Reader using Illumina BeadScan image data acquisition software.
Microarray data analysis
Expression data were quantile normalized and log2 transformed using the R-package lumi
. 1,342 unique probes that hybridized to transcript sequences containing a strain-specific polymorphism or an indel (insertion/deletion) were excluded from further gene expression-related analysis. Sequence variations between AKR and DBA/2 (SNPs and insertions-deletions) were obtained from sequencing data generated by the Mouse Genomes Project: http://www.sanger.ac.uk/resources/mouse/genomes/
. Probes were also removed if no sample had a detection p-value less than 0.01. 9,316 and 11,919 probes remained for further analysis in experiments 1 and 2, respectively. To determine strain effects on the transcriptome, only the results of unloaded bone marrow macrophages were analyzed. Log2 fold change, unadjusted p-values, and false discovery rate adjusted p-values were determined separately for the two independent datasets with the limma
package in R 
using a fitted linear model with the following equation: Yi
Strain-Loading, with the strain and loading βs as additive covariates and the strain-loading interaction β as an interactive covariate.
Gene set enrichment analysis was performed for all expressed transcripts to identify possible pathways altered by strain, loading and strain-loading interactions. The romer
function in the limma
package was used to test for gene set enrichment analysis 
with the Molecular Signature Database from the Broad Institute as gene sets 
. We present and discuss further only the pathways that were significantly enriched using the permutation test p-value threshold of 1.0E-04. The MIAME compliant microarray dataset from this study is available through the Gene Expression Omnibus server, accession number GSE38736.
Real-Time quantitative PCR (qPCR)
To validate the findings of gene transcript measurements by microarray, we assessed the expression of selected gene transcripts by using qPCR approach. The expression levels of tribbles homolog 3 (Trib3
), activating transcription factor 4 (Atf4
), ceroid-lipofuscinosis neuronal 5 (Cln5
), and ATP-binding cassette sub-family G member 1 (Abcg1
) were quantified relative to the endogenous control gene, ubiquitin C (Ubc
) using pre-designed TaqMan gene expression assays (Applied Biosystems). Mean fold changes for each sample were calculated as previously described 
Unloaded and cholesterol loaded BMM from AKR and DBA/2 were lysed with 0.5 mL of M-PER mammalian protein extraction reagent (Pierce Biotechnology). Cell lysates (45 µg protein/lane) were loaded, separated on a 4–15% gradient polyacrylamide gel and transferred to PVDF membranes by semi-dry electroblotting. After blocking with rapid blocking buffer (Amresco, Cat # M325) for 1 hour, the membrane was incubated overnight with rabbit antibody to TRIB3 (Proteintech, Cat # 13300) at 4°C. The membrane was washed and further incubated with HRP goat anti-rabbit IgG (Amresco, Cat # N791) secondary antibody for 1 hour. The membrane was then exposed to an enhanced chemiluminescent system, and bands were visualized by exposure to X-ray film. After stripping, GAPDH protein was visualized as loading control using goat antibody to GAPDH (Abcam, Cat # ab9483), followed by HRP rabbit anti-goat IgG as described above. TRIB3 band density was quantified using Total Lab TL120 software (Nonlinear Dynamics) and normalized to the respective GAPDH band density.