Fig. 6

Thrombin induces the downregulation of ACSL4 after acute cerebral ischemia/reperfusion. a ACSL4 protein level was examined in N27 cells treated with thrombin (0.5 U/mL) for 24 h. Western blots were analyzed with Image J and normalized to β-actin expression. Data are means ± SEM, n = 4. t test was performed. b ACSL4 protein levels were examined from the ischemic ipsilateral hippocampus of rats that underwent MCAO after 0, 6, or 24 h of reperfusion. Western blots were analyzed with Image J and normalized to β-actin expression. Data are means ± SEM, n = 3 animals per group. One-way ANOVA with post-hoc Tukey test was performed. c Degeneration of CA1 pyramidal neurons following I/R. Hippocampal sections from sham, R3h (3 h after MCAO/R), and R6h (6 h after MCAO/R) rats were stained with NeuN (red) and Fluoro-Jade (green). Representative Immunofluorescence staining for ACSL4 from adjacent brain tissue sections spaced 4 μm apart. d Quantification of survival and death neuron numbers in a region of interest (ROI = 0.1 mm²) Data are means ± SEM, n = 3. Two-way ANOVA with post-hoc Tukey test was performed. e The intensity of ACSL4 immunofluorescence in an ROI was quantified using Image J. Data are means ± SEM, n = 3 animals per group. One-way ANOVA with post-hoc Tukey test was performed. f Thrombin protein levels were examined from the ischemic ipsilateral hippocampus of rats 0.5, 1, and 1.5 h after MCAO. Western blots were analyzed with Image J and normalized to β-actin expression. Data are means ± SEM, n = 3 animals per group. One-way ANOVA with post-hoc Tukey test was performed. g Cell viability of WT, ACSL4 KO, and ACSL4 OE N27 cells 24 h after thrombin (0.5 U/mL) treatment. Data are means ± SEM, n = 6 wells from one representative of 3 independent experiments. One-way ANOVA with post-hoc Tukey test was performed