Fig. 15.

Inhibition of PKDs suppresses the LPA-induced pro-inflammatory phenotype. a Serum-starved BV-2 cells were treated with DMSO, DMSO plus LPA (1 μM), and LPA (1 μM) in the presence of CRT (1 μM) for 24 and 48 h. Cell lysates were collected, and expression of COX-2 and Arg-1 was analyzed by western blotting. One representative blot and the densitometric analysis (mean + SD) from three independent experiments are presented. b BV-2 cells were stained with PE-conjugated anti-CD40 (upper panel), APC-conjugated anti-CD86 (middle panel), or PE-conjugated anti-CD206 (lower panel) antibodies and analyzed using a Guava easyCyte 8 Millipore flow cytometer. Results from four independent experiments performed in triplicate are shown as mean value + SD. c Intracellular ROS levels generated in response to LPA by BV-2 cells were determined. Serum-starved cells were incubated with carboxy-H₂DCFDA and treated with DMSO, DMSO plus LPA (1 μM), or LPA (1 μM) plus CRT (1 μM), and the fluorescence intensity was evaluated. Results (three independent experiments performed in triplicate) are presented as mean values + SD. d Serum-starved BV-2 cells were incubated with DMSO, DMSO plus LPA (1 μM), or LPA (1 μM) plus each inhibitor for the indicated times, and the production of NO was determined by measuring the total nitrate concentration in the supernatants. Data (two separate experiments performed in triplicate) are presented as mean values + SD. e CATH.a neurons were incubated for 24 h with conditioned media collected from DMSO or LPA-treated BV-2 cells cultured in the absence or presence of CRT (1 μM) for 24 h. LDH activity was determined in the neuronal supernatants after 24 h. Cytotoxicity was calculated according to the manufacturer’s instructions (*p < 0.05, **p < 0.01, ***p < 0.001, compared to DMSO-treated cells; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, each inhibitor compared to LPA-treated cells; one-way ANOVA with the Bonferroni correction)