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Figure 2. Effect of the silencing of GM3 synthase on glutamate-induced neuronal cell death. After mock and siST3GalV cells were treated with or without 5 mM glutamate for 8 h, the following experiments were performed. A) Cells were analyzed for morphological changes by phase-contrast microscopy. B) Cell viability was assayed by Cell Counting Kit-8. Data are the means ± SD of at least three independent experiments. *P < 0.001 vs. mock control; #P < 0.001 vs. glutamate-treated mock. C) After incubation with H2DCFDA for 20 min, their relative ROS generation was measured by flow cytometry. Data are means ± SD of at least three independent experiments. *P < 0.005 vs. mock control; #P < 0.005 vs. glutamate-treated mock. D) Calcium ion (Fluo-4 fluorescence) changes were analyzed by flow cytometry. HT22 cells were treated with 20 µM cobalt chloride (Cobalt) in the presence or absence of 5 mM glutamate for the inhibition of Ca2+ influx for 8 h, or with 10 µM BAPTA for normalization for 20 min. Data are means ± SD of five independent experiments. *P < 0.0001 vs. mock control; #P < 0.0001 vs. glutamate-treated mock. E) Intracellular GSH was measured by total GSH quantification kit. The amounts of GSH were evaluated by GSH standard curve analysis. Means ± SD (n=3). *P < 0.01 vs. mock control; #P < 0.01 vs. siST3GalV control. F) Cells were collected, extracted, and measured for 12-lipoxygenase activity (12-HETE production) by using a correlated enzyme-linked immunoassay kit. Means ± SD (n=3). *P < 0.005 vs. mock control; #P < 0.05 vs. glutamate-treated mock.
