Figure 5

Verification of proteomic results by flow cytometry and qPCR. Cell surface expression of ICAM1/CD54 and SIGLEC1/CD169 on CD14⁺ monocytes (A) and THP-1 cells (B). (A) Monocytes were either left untreated or stimulated for 24 h or 48 h with 50 ng/ml LPS. White graphs: isotype controls, in light grey: expression at the indicated time point of unstimulated controls, in dark grey: expression at the indicated time points after LPS treatment. The data shown are representative of two different donors independently analyzed. Depicted are the intensity levels of the indicated proteins expressed on the CD14⁺ cell population (B) THP-1 cells were either left untreated or stimulated with 50 ng/ml LPS for 4 h, 24 h, and 48 h. White graphs: expression in unstimulated controls at t = (0), in light grey expression at the indicated time point of unstimulated controls, in dark grey: expression at the indicated time points after LPS treatment. The data shown are representative of three independent experiments. (C,D) Gene expression changes of selected glycoproteins induced by LPS treatment revealed by qPCR. (C) Monocyte mRNA expression changes of GPR84, MMP9, LAMP3, DPEP2, and ITGB8 at different time points after LPS treatment of naïve (pre: no), or 24 h LPS pre-stimulated (pre: LPS) monocytes re-stimulated with LPS for the indicated time points. (D) THP-1 mRNA expression changes of GPR84, MMP9, IL4i1, EBI3, and STEAP4 at different time points after LPS treatment of naïve (pre: no), or 24 h LPS pre-stimulated (pre: LPS) THP-1 cells re-stimulated with LPS for the indicated time points. (C + D) Plotted are fold changes normalized to the house-keeping gene PPIB (monocytes) or HPRT (THP-1) and compared to naïve unstimulated control cells at t = (0). Samples (mean of three independent experiments (THP-1 cells) or three independent donors (monocytes) ± SE) *p < 0.05, **p < 0.01, ***p < 0.001.