A. qPCR validation of select genes from overlapping set (from Table 1); B. Expression of lipogenesis genes and transcription factors in olr1 KO mice. White bars – microarray data; black bars – qPCR data. All P values<0.05.

A. Wound healing assay. Upper panel - representative images of wound healing assay performed using HCC1143 cells transfected with either empty plasmid or OLR1 cDNA vector; Lower panel – graph depicting the distance between edges of the wound after 36 hours of incubation. (*) – p<0.01; B. Adhesion assay. Upper panel- representative images of adherent non-transfected HCC1143 cells loaded with CellTracker Red CMTX (Invitrogen, Carlbad, CA) and applied to non-activated or activated (50 µg/ml oxLDL, 4 hrs) confluent HUVECs transfected with OLR1 Silencer or scrambled siRNA. Lower panel - graph depicting the number of adherent cells averaged from multiple fields of view in triplicate cultures. (*) – p<0.05 compared to non-activated control (“scrRNA”); (†) - p<0.05 compared to scrambled RNA; C. Colorimetric transendothelial migration assay. Upper panel – verification of the confluence of HUVECs on the membranes by staining cells with CellTracker Red CMTX. Lower panel – absorbance values of stain extracted from the cells migrated through TNFα-activated endothelial monolayer in presence of OLR1 neutralizing antibody or human IgG (Control).

A diagram depicting a set of overlapping genes between transformation and Olr1 KO transcriptomes. From the set of 238 genes upregulated during transformation, 26 genes were found to be inhibited in Olr1 KO mice. Vast majority of these genes carried NF-κB sites in their proximal promoter sequences. In total, 86 NF-κB target genes were found to be inhibited in Olr1 KO mice with enrichment for regulation of apoptosis (p = 0.0002), proliferation (p = 0.00003), wound healing (p = 0.0002), defense response (p = 0.0011), immune response (p = 0.0003) and cell migration (p = 0.0009).

These cells were transfected with either empty vector or OLR1 cDNA (Origene, Rockville, MD) using Lipofectamine 2000 (Invitrogen). Transfection efficiency (70–80%) was evaluated using GFP vector. RNA was extracted 48 hours post-transfection, converted into cDNA and the expression of genes was determined by quantitative PCR. A. Efficiency of transfection (cells transfected with GFP vector). B. Quantitative PCR plot. Note the enhancement of OLR1 expression in both control and OLR1-transfected cultures in response to ox-LDL. C. Expression of genes involved in apoptosis and proliferation. In order to stimulate OLR1 associated signaling requiring OLR1-ligand interaction, OLR1 transfected cells were treated with 40 µg/ml ox-LDL for 24 hours; graphs represent comparison with untreated control cells transfected with empty vector; D. Basal expression of OLR1, PLA2G4B and lipogenesis genes in normal human mammary epithelial cells (MCF10A) and breast cancer cells (HCC1143); E. Expression of OLR1, PLA2G4B and lipogenesis genes in MCF10a and HCC1143 cells transfected with OLR1 treated according to the protocol described above. F. Expression of lipogenesis transcription factors in MCF10a and HCC1143 cells transfected with OLR1 and treated according to the protocol described above. All experiments were conducted in triplicates. (*) p<0.05 compared to respective control; (†) – p<0.05 compared to MCF10A.