Interaction of Apoptotic Cells with Macrophages Upregulates COX-2/PGE2 and HGF Expression via a Positive Feedback Loop
Figure 3
Inhibition of COX-2 downregulates HGF expression. RAW 264.7 cells were pretreated with NS-398 or indomethacin (Indo) for 1 h and then stimulated with apoptotic Jurkat cells (ApoJ) for 2 h to detect HGF mRNA expression (a, b) or for 24 h to detect secreted HGF (c). RAW cells were transfected with COX-2 or control vehicle (siRNA-GFP) for 6 h and then incubated with ApoJ for 2 (d, f) or 24 h (e, g). (h, i) RAW cells were transfected with COX-1 siRNA or control vehicle (siRNA-GFP) for 48 h and then incubated with ApoJ for 24 h. (a, b, c) HGF mRNA levels were analyzed using semiquantitative RT-PCR and normalized to -actin mRNA levels. (d, e, h) Immunoblots of total cell lysates were performed with anti-COX-2 or COX-1 antibodies. (c, g, i) HGF levels in conditioned media were measured by ELISA. Values represent mean ± SEM of three or more separate experiments; .
