Figure 4. Effects of LPA receptors on the neuroprotective functions of cPA and LPA against CoCl₂-induced apoptosis.

(A) Expression of LPA receptors in Neuro2A cells. Total RNA was extracted from Neuro2A cells, and the expression level of each LPA receptor was determined by quantitative real-time PCR. The expression levels were normalized to those of LPA₁ and expressed in terms of the mean ± SE values. (B) The effects of Ki16425 on the neuroprotective functions of cPA and LPA against CoCl₂-induced apoptosis of Neuro2A cells. Neuro2A cells were pretreated with or without 10 µM Ki16425 for 20 min. Subsequently, the cells were incubated with 300 µM CoCl₂ in the presence of 10 µM cPA or LPA for 24 hours. The cells were then stained with FITC-Annexin V and subjected to flow cytometric analysis. The data represent the mean ± SE values from triplicate independent experiments (***P<0.001 vs. the CoCl₂-treated group). (C) Expression of LPA₂ in Neuro2A cells. Neuro2A cells were transfected with siRNA against LPA₂ or non-target siRNA. Total RNA was extracted from each transfected Neuro2A cell, and the expression level of each LPA receptor was determined by quantitative real-time PCR. The expression levels of LPA₂ was normalized to those of Neuro2A cells transfected with non-target siRNA. The resulting data represent the mean ± SE values (***P<0.001 vs. the mock group). (D) The effects of LPA₂ knockdown on the neuroprotective effects of cPA and LPA against CoCl₂-induced apoptosis of Neuro2A cells. Neuro2A cells transfected with either siRNA against LPA₂ or non-target siRNA were incubated with 300 µM CoCl₂ in the presence of 10 µM cPA or LPA for 24 hours. Cells stained with FITC-Annexin V were subjected to flow cytometric analysis. The data represent the mean ± SE values from triplicate independent experiments (*P<0.05, ***P<0.001 vs. the CoCl₂-treated group; n.s., not significant).