Fig. 4. S1P signalling pathway involves the activation of RhoA and LIM kinase, a degradation target. (A) S1P- and LPA-induced retinal growth cone collapse are blocked by RhoA kinase inhibitor (Y-27632). Y-27632 was added immediately prior to the addition of S1P in stage 32 retinal explants cultured for 24 hours. ^*P<0.05, Mann-Whitney U test. (B,C) Cumulative distribution of turning angles after Y-27632 treatment. The repulsive response elicited by S1P is inhibited by application of Y-27632 prior to the start of the turning assay. (C) Mean turning graph from data in B. ^***P<0.005, Kolmogorov-Smirnov test. (D) Quantitative immunofluorescence analysis of LIMK-P and LIMK immunoreactivities. A 2-minute S1P treatment caused a significant increase in LIMK-P, whereas LIMK signal remained unchanged. LIMK-P signal was not affected by a 2-minute LPA treatment. At 5 minutes, S1P induced a significant decrease of both LIMK-P and LIMK immunoreactivities within growth cones when compared with control. This decrease is abolished by lactacystin treatment. (E) Representative pictures of LIM kinase-P (LIMK-P) and LIM kinase (LIMK) immunoreactivities within growth cones treated with S1P for 2 or 5 minutes, or when lactacystin was applied 10 minutes before S1P application. Numbers inside bars indicate growth cones tested. ^**P<0.01, ^***P<0.005, Mann-Whitney U test. Scale bar in D: 10 µm.