Fig 1: Different concentrations of erastin induce ferroptosis and upregulate miR-494-3p in the LUHMES cells. LUHMES cells were stimulated by 0, 0.25, 0.5, 1, 2, and 4 µM erastin for 24 hours. (a) qRT-PCR analysis of miR-494-3p in erastin-stimulated LUHMES cells. (b) Cell proliferation analysis by CCK-8 assay. (c) The levels of TH, NSE, ACSL4, and GPX4 were estimated by western blotting in erastin-stimulated LUHMES cells. (d) Relative protein levels of TH, NSE, ACSL4, and GPX4 based on the western blotting results. Results were representative data from triplicate experiments, and data are means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2: The impact of miR-494-3p on viability, ROS, ferroptosis, neuron injury, and REST in erastin-induced LUHMES cells. Erastin-stimulated LUHMES cells were transfected with miR-494-3p inhibitor, miR-494-3p mimics, or NC or treated with ferroptosis inhibitor (ferrostatin-1). (a) The miR-494-3p level was tested by qRT-PCR assay in each group. (b) Cell proliferation was monitored via CCK-8 assay in the above groups. (c and d) ROS activity was identified via a flow cytometer in each group. (e) TEM images showing mitochondrial morphology in each group. Scale bar = 2 µm. Mitochondria are marked with blue arrows. (f) Western blotting analysis of TH, NSE, ACSL4, and GPX4 in each group. (g and h) qRT-PCR and western blotting analysis of REST. (i) WT and Mut REST 3'-UTR plasmids were constructed and cotransfected with miR-494-3p mimics into cells, and then, the luciferase activity was tested by dual-luciferase reporter assay. Results were representative data from triplicate experiments, and data are means ± SD. *P < 0.05 and ***P < 0.001 vs. control or NC group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. erastin+NC group; &P < 0.05 and &&P < 0.01 vs. erastin group.
Fig 3: REST attenuates the effect of miR-494-3p on viability, ROS, ferroptosis, and neuron injury in erastin-induced LUHMES cells. Erastin-stimulated LUHMES were cotransfected with miR-494-3p inhibitor and sh-REST or REST-overexpressed plasmid and miR-494-3p mimics, respectively. (a and b) qRT-PCR showing the levels of miR-494-3p (a) and REST (b). (c) Cell viability was measured through the CCK-8 assay. (d) A Flow cytometer verified the change in ROS activity. (e) TEM images showing changes in mitochondrial morphology. Scale bar = 2 µm. Mitochondria are marked with blue arrows. (f and g) Immunofluorescence images showing ACSL4 (f) and TH (g) expressions. Magnification: 200×, scale bar = 100 µm. (h) Western blotting analysis showing REST, TH, NSE, ACSL4, and GPX4 levels. Results were representative data from triplicate experiments, and data are means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. inhibitor+sh-NC group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. mimics+vector group.
Fig 4: Meg3 regulated GPX4 via p53. A,B) RT-qPCR analysis showed that the overexpression and knockdown of p53 had no impact on FTH1 and ACSL4 expressions. C) The impact of p53 overexpression and knockdown on GPX4 expression was evaluated using RTqPCR analysis. D,E) Western blot images and quantitative analysis showed the effect of p53 overexpression and knockdown on GPX4 protein expression. F,G) Western blot images and quantitative analysis showed that the overexpression of p53 also reversed the impact of si-Meg3 on the protein expression of GPX4. H,I) Chip assay suggested that p53 significantly bound to GPX4 promoter in injury elicited by OGD + HG; anti-histone-3 (a-H3) served as a positive control, and IgG was used as a negative control. Data were expressed as the mean ± SD. The experiments were carried out for three times. *p<0.05 vs the OGD-HG+ Meg3-NC group; $p<0.05 vs the OGDHG+ Meg3-siRNA group; #p<0.05 vs the Meg3-NC group.
Fig 5: Effects of REST on viability, ROS, ferroptosis, and neuron injury in erastin-induced LUHMES. After erastin stimulation, LUHMES were transfected with REST-overexpressed plasmid or sh-REST. (a) The levels of REST were evaluated through qRT-PCR analysis. (b) CCK-8 analyzed the impact of REST on the viability of cells. (c and d) Flow cytometry analyzed the impact of REST on ROS activity in the treated LUHMES cells. (e) TEM shows mitochondrial morphology. Scale bar = 2 µm. Mitochondria are marked with blue arrows. (f) Western blotting analysis of REST, TH, NSE, ACSL4, and GPX4, and quantitative analysis of these proteins based on the western blotting results. Results were representative data from triplicate experiments, and data are means ± SD. ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. erastin+vector group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 vs. erastin+sh-NC group.
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