Figure 6. RhoA-dependent HGF secretion from RAW 264.7 cells and exogenous PGE₂, PGD₂, and HGF mediate EMT reduction in LA-4 cells.

(a,b,d) RAW 264.7 cells were pretreated with 30 μM Y-27632 for 1 h and then stimulated with apoptotic Jurkat cells (ApoJ) for 20 h. (e) RAW cells were transfected with RhoA siRNA or control vehicle (siRNA-GFP) for 24 h, then incubated with ApoJ for 20 h. Conditioned medium (CM) was added to LA-4 cells in the presence of TGF-β1 for 48 h. (a,c,d) RAW 264.7 cells were stimulated with ApoJ for 20 h. CM was added to LA-4 cells in the presence of TGF-β1 with or without the antagonist of c-Met (250 nM PHA-665752). (f) PGE₂ (50 and 150 pg/ml), PGD₂ (7 and 17 pg/ml), or HGF (150 and 400 pg/ml) was added to LA-4 cell culture in the presence of TGF-β1 for 48 h. (a) Morphological changes in the cells were examined by phase-contrast microscopy (Scale bars = 50 μm). (b,c,e,f) Immunoblots of total cell lysates were performed with anti-E-cadherin, N-cadherin, or α-SMA antibodies. Right: Densitometric analysis of the indicated EMT markers’ relative abundances. (d) The amounts of Snail1/2, Zeb1/2, and Twist1 mRNAs in LA-4 cell samples were analyzed by real-time PCR and normalized to that of Hprt mRNA. Values represent the mean ± s.e.m. of three independent experiments. *P < 0.05 compared with control; ⁺P < 0.05 as indicated.