Research Article

Bone Components Downregulate Expression of Toll-Like Receptor 4 on the Surface of Human Monocytic U937 Cells: A Cell Model for Postfracture Immune Dysfunction

Figure 4

Involvement of glycoprotein 96 (gp96) but not protein associated with toll-like receptor 4 (PRAT4A) in the downregulation of TLR4 surface expression in U937 cells after bone component (BC) exposure. (a) U937 cells were pretreated with 100 μM chloroquine for 2 h before 50 ng/mL BC treatment for 24 h. Membranous portion of TLR4 in U937 cells was examined using flow cytometry, and results are presented as percentages of the control. Data are presented as mean ± SEM (); compared with the control group. (b) U937 cells were treated with 50 ng/mL BC for 24 h. Cellular gp96 and PRAT4A in U937 cells were detected using western blot analysis. β-actin was used as an internal control. Data represent the results of 3 independent experiments (mean ± SEM; was considered significant). (c) U937 cells were stimulated with 50 ng/mL BC for 24 h. The gp96, PRAT4A, and TLR4 proteins were identified with specific antibodies by immunofluorescence. 4′,6-Diamidino-2-phenylindole was used to characterize the nucleus. The slides were observed by confocal microscopy.
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