Figure 1.
Treatment with CoCl2 induces apoptosis in Neuro2A cells.
(A) Effects of CoCl2 on the viability of Neuro2A cells. Neuro2A cells were incubated with various concentrations of CoCl2 for 24 hours. Cell viability was estimated as described in the materials and methods section. The data represent the mean ± SE values from triplicate independent experiments. (B) Generation of reactive oxygen species (ROS) induced by CoCl2. Neuro2A cells were incubated with 300 µM CoCl2, and ROS generation was measured after 5 and 15 min. The data represent the mean ± SE values from triplicate independent experiments (*P<0.05, ***P<0.001 vs. the CoCl2-treated group). (C) Morphologic changes in the nuclei of CoCl2-treated Neuro2A cells. Neuro2A cells incubated in the absence (left) or presence (right) of 300 µM CoCl2 for 24 hours were fixed and stained with DAPI and examined by fluorescence microscopy. (D) Analysis of apoptosis-associated PS exposure on CoCl2-treated Neuro2A cells by using FITC-Annexin V. Neuro2A cells were treated with (right) or without (left) 300 µM CoCl2 for 24 hours and stained with FITC-Annexin V. The population of FITC-Annexin V-positive Neuro2A cells was quantified by flow cytometry.
Figure 2.
cPA protects against CoCl2-induced apoptosis in Neuro2A cells.
(A) The effects of cPA and LPA on a number of adhesive Neuro2A cells treated with CoCl2. Neuro2A cells were incubated with 300 µM CoCl2 in the presence of 10 µM cPA or LPA for 24 hours, and the number of cells attached to the surface of dishes was determined (*P<0.05, **P<0.01 vs. the CoCl2-treated group). (B & C) Analysis of the percent of FITC-Annexin V- and FITC-DEVD-FMK-positive Neuro2A cells after CoCl2 treatment by flow cytometry. Neuro2A cells were incubated with 300 µM CoCl2 and various concentrations of either cPA or LPA for 24 hours. Cells were subsequently stained with either FITC-Annexin V (B) or FITC-DEVD-FMK, an activated caspase-3 inhibitor (C) and subjected to flow cytometric analysis. The data represent the mean ± SE values from triplicate independent experiments (*P<0.05, **P<0.01, ***P<0.001 vs. the CoCl2-treated group).
Figure 3.
Effects of cPA and LPA on the expression of the Bcl-2 protein family.
(A) Neuro2A cells were incubated with 300 µM CoCl2 in the presence of 10 µM cPA or LPA for the time indicated. Protein levels of Bax, Bcl-2, and β-actin were determined by western blot analysis. (B) The expression of Bax and Bcl-2 was determined using a densitometer, and the Bax/Bcl-2 ratio was calculated and normalized to cells without any treatment. The data represent the mean ± SE values from triplicate independent experiments (***P<0.001 vs. CoCl2-treated group).
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).