Figure 1

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).