Figure 5.

Effect of CD9 silencing on the shedding of LR11 in THP-1 cells. (a) Effect of the metalloproteinase inhibitor GM6001 on the shedding of LR11 in non-adherent hematopoietic cells. PMA-stimulated (20 ng ml⁻¹, 24 h) THP-1 and U937 cells were incubated with or without GM6001 (50 μM) for 24 h. The protein levels of sLR11 released from these cells were analyzed by western blot analysis. (b) The effect of shRNA-mediated CD9 silencing in THP-1 cells. THP-1 cells stably transfected with the CD9-targeted shRNA or with control shRNA (mock control) were generated as described in Materials and methods. The cell surface expression levels of CD9 in the CD9 shRNA-silenced-THP-1 cells (clone #8; dashed line) and mock controls (clone #1; solid line) with or without the stimulation of PMA (20 ng ml⁻¹, 24 h) were evaluated by flow cytometry (upper panel). Gray-colored histograms represent the cells labeled with an isotype control antibody. Cells were either unstimulated or stimulated for 24 h with 20 ng ml⁻¹ PMA in the presence or absence of GM6001 (50 μM). The protein levels of sLR11 released from the cells were analyzed by western blot analysis. In all cases, the cells were cultured with fresh serum-free media. Data are shown as the fold increase relative to the control and presented as the mean±s.d. (error bars, n=3). *P<0.05; ns, not significant; ND, not detected.