Fig 2: Comparison of the binding of transcription factors (NF-?B, PU.1 and ATF2) to the TREM-1 promoter regions 1 and 2 between low ESS and no flow conditions. 3D co-cultures of HCAECs, HCASMCs, and monocytes (THP-1) were prepared on the coverslips and cultured overnight. Cells were then exposed to low ESS or no flow for or 30 min. ChIP assay was then performed as described in the “Methods”. ESS: endothelial shear stress, TREM-1: triggering receptor expressed on myeloid cells-1, NF-??: Nuclear factor kappa B, ATF2: Activating transcription factor 2.

Fig 3: Effect of TREM-1 suppression by siRNA on the mRNA expression of pro-inflammatory molecules and matrix-degrading enzymes in 3D co-cultures exposed to low shear stress. HCAECs, HCASMCs, and THP-1 cells were transfected with control-siRNA or TREM-1-siRNA for 6 h. The 3D co-cultures were prepared on the coverslips and cultured overnight. Cells were then exposed to low ESS or no flow for 1 h followed by additional culture for 1 h. Real time RT-PCR for the quantification of mRNA was performed as described in the “Methods”. IL: interleukin, MMP: matrix metalloproteinase, MCP-1: monocyte chemoattractant protein-1, TREM-1: triggering receptor expressed on myeloid cells-1, HCAECs: human coronary artery endothelial cells, HCASMCs: human coronary artery smooth muscle cells. Data were averaged over 3 separate experiments.

Fig 4: Suppression of TREM-1 protein expression by siRNA and its effect on the expression of matrix-degrading enzymes. (A) Suppression of TREM-1 expression by siRNA in monolayer culture of HCAECs. HCAECs were transfected with control-siRNA or TREM-1-siRNA as described in the “Methods”. Cells were then plated on coverslips and exposed to low ESS or no flow for 1 h followed by additional culture for 5 h. Cells were then fixed and immunofluorescence staining for TREM-1 was performed as described in the “Methods”. (B) Suppression of cathepsin S and MMP-1 protein expression in 3D co-cultures transfected with TREM-1 siRNA. HCAECs, HCASMCs and THP-1 cells were transfected with control-siRNA or TREM-1-siRNA for 6 h prior to preparing the 3D co-culture on coverslips. The cells were then exposed to low ESS for 1 h followed by additional culture for 5 h. Immunofluorescence staining for cathepsin S and MMP-1 were performed as described in the “Methods”. TREM-1: triggering receptor expressed on myeloid cells-1, siRNA: small interfering RNA, ESS: endothelial shear stress, MMP-1: matrix metalloproteinase-1, HCAECs: human coronary artery endothelial cells, HCASMCs: human coronary artery smooth muscle cells.

Fig 5: Effect of various shear stress conditions on the TREM-1 mRNA expression and protein synthesis in co-culture model. (A) Expression of TREM-1 mRNA in 3D co-culture cells exposed to low ESS, medium ESS, high ESS, and no flow (control). 3D co-culture of HCASMCs, HCAECs and monocytes (THP-1) cells were prepared on the cover slips and exposed to low ESS, medium ESS, high ESS, or no flow (control) for 1 h followed by additional culture for 1 h. Total RNA was extracted and expression of mRNA was quantified by real time RT-PCR as described in the “Methods”. (B) Immunoblotting of membrane-bound TREM-1 in 3D co-culture cells exposed to low ESS or no flow (control). 3D co-culture of HCASMCs, HCAECs and monocytes (THP-1) cells were prepared on the cover slips and exposed to low ESS or no flow (control) for 1 h followed by additional culture for 5 h. Total proteins of cell lysates were subjected to electrophoresis (10% SDS-12.5% PAGE gel) and immunoblotting to TREM-1 and ß-actin as described in the “Methods” (B presents the blots at 5 h. The original blots at 5 and 24 h are presented in the Supplemental Fig. S3). (C) Immunofluorescence staining of TREM-1 in 3D co-culture cells exposed to low ESS, medium ESS, high ESS, and no flow (control). 3D co-culture of HCASMCs, HCAECs and monocytes (THP-1) cells were prepared on the cover slips and treated with low ESS, medium ESS, high ESS, or no flow (control) for 1 h followed by additional culture for 5 h. Immunofluorescence staining to TREM-1 was then performed as described in the “Methods”. ESS: endothelial shear stress. TREM-1: triggering receptor expressed on myeloid cells-1. Data were averaged over 3 separate experiments (A).