Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: Exosome carried NEAT1 into the cerebral cortex and NEAT1 was significantly highly expressed in sepsis-induced ferroptosis A Brain vascular permeability was detected by EBD leakage in control and model rats ( n = 10). Extracted dye contents in the formamide extracts were quantified at 620 nm; B ROS level in cerebral cortex homogenate was detected by flow cytometry. C – E ELISA analysis on Fe ion, GSH, and GPX4 levels. F HE staining on cerebral cortex in control and model rats. G Western blot analysis on serous exosome biomarkers of TSG01, CD9, and CD63. H , I The expression of NEAT1 was detected by qRT-PCR in the serous exosome (normalized to synthetic cel-miR-39-3p) and cerebral cortex (normalized to GAPDH), respectively. J The correlation analysis on the expression of NEAT1 in exosome and cerebral cortex. ** P < 0.01 vs Control; *** P < 0.001 vs Control; EBD, Evans blue dye.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Permeability, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: miR-9-5p, sponged by NEAT1, overexpressed in vitro and in vivo and might promote cell apoptosis in sepsis-induced ferroptosis by targeting TFRC and GOT1 A The binding regions between miR-9-5p with sequences from NEAT1, TFRC , and GOT1 . B , C The expression level of miR-9-5p, TFRC , and GOT1 in model and control was tested by qRT-PCR in vivo (normalized to U6 or GAPDH ). *** P < 0.001 vs control. D The protein levels of TFRC and GOT1 in cerebral cortex were assessed using Western blotting. E The protein level of TFRC in cerebral cortex was showed by immunohistochemistry. F , G The dual-luciferase reporter gene assay analyzes the binding relationship between miR-9-5p with sequences from NEAT1, TFRC , and GOT1 . *** P < 0.001 vs NC. H , I The expression level of miR-9-5p, TFRC , and GOT1 in iron-rich (100 μM FeCl 3 ), control (100 μM FeCl 3 + control serum), and model (100 μM FeCl 3 + sepsis serum) group was tested by qRT-PCR (normalized to U6 or GAPDH ). * P < 0.05, ** P < 0.01, *** P < 0.001 vs iron-rich and control. J The protein levels of TFRC and GOT1 assessed using western blotting in vitro. K , L The immunofluorescence for detecting TFRC and GOT1 (blue, DAPI; green, TFRC; red, GOT1).

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: In Vitro, In Vivo, Binding Assay, Expressing, Control, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Luciferase, Reporter Gene Assay, Immunofluorescence

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: Increased miR-9-5p regulates the expression of TFRC and GOT1 and relieves ferroptosis in vitro The bEnd.3 cells were transfected with miR-9-5p angomir, followed by serum stimulation. A The expression levels of NEAT1, miR-9-5p, TFRC , and GOT1 in control, model, and model + miR-9-5p angomir were tested by qRT-PCR (normalized to U6 or GAPDH ). B The sequence for NEAT1 mutant. C The protein levels of TFRC and GOT1. D , E The immunofluorescence for TFRC and GOT1 (blue, DAPI; green, TFRC; red, GOT1). F The cell viability of bEnd.3 cells were evaluated by CCK-8 assay. G – K The levels of ROS, Fe ion (Fe 2+ and Fe 3+ ), GSH, GPX4, and MDA were assessed. * P < 0.05, ** P < 0.01, *** P < 0.001 vs control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs model.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vitro, Transfection, Control, Quantitative RT-PCR, Sequencing, Mutagenesis, Immunofluorescence, CCK-8 Assay

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: Enhanced NEAT1 promoted ferroptosis and increased the expression of TFRC and GOT1 in vitro NEAT1 was overexpressed in bEnd.3 cells. A The expression levels of NEAT1, miR-9-5p, TFRC , and GOT1 in Vector, WT NEAT1 (wild NEAT1 sequence), and MUT NEAT1 (site-directed mutant NEAT1) by qRT-PCR (normalized to U6 or GAPDH ). B The protein levels of TFRC and GOT1 showed by western blotting. C , D The immunofluorescence for TFRC and GOT1 (blue, DAPI; green, TFRC; red, GOT1). E The cell viability of cells detected by CCK-8 assay. F – J The levels of ROS, Fe ion (Fe 2+ and Fe 3+ ), GSH, GPX4, and MDA. ** P < 0.01, ** P < 0.01, *** P < 0.001 vs Vector; ## P < 0.01, ### P < 0.001 vs WT NEAT1.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vitro, Plasmid Preparation, Sequencing, Mutagenesis, Quantitative RT-PCR, Western Blot, Immunofluorescence, CCK-8 Assay

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: miR-9-5p suppressed the expression of TFRC and GOT1 in vivo A – D The qRT-PCR analysis on the expression of NEAT1, miR-9-5p, TFRC , and GOT1 in the cerebral cortex of control, model, and model + miR-9-5p angomir rats, respectively (normalized to U6 or GAPDH ). E The western blotting analysis on TFRC and GOT1. F The protein level of TFRC in four brain regions were assessed by Immunohistochemistry. *** P < 0.001 vs control; ### P < 0.001 vs model.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vivo, Quantitative RT-PCR, Control, Western Blot, Immunohistochemistry

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: miR-9-5p suppressed the expression of and GOT1 in vivo The protein level of GOT1 in four brain regions was assessed by Immunohistochemistry.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vivo, Immunohistochemistry

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: Exosome carried NEAT1 into the cerebral cortex and NEAT1 was significantly highly expressed in sepsis-induced ferroptosis A Brain vascular permeability was detected by EBD leakage in control and model rats ( n = 10). Extracted dye contents in the formamide extracts were quantified at 620 nm; B ROS level in cerebral cortex homogenate was detected by flow cytometry. C – E ELISA analysis on Fe ion, GSH, and GPX4 levels. F HE staining on cerebral cortex in control and model rats. G Western blot analysis on serous exosome biomarkers of TSG01, CD9, and CD63. H , I The expression of NEAT1 was detected by qRT-PCR in the serous exosome (normalized to synthetic cel-miR-39-3p) and cerebral cortex (normalized to GAPDH), respectively. J The correlation analysis on the expression of NEAT1 in exosome and cerebral cortex. ** P < 0.01 vs Control; *** P < 0.001 vs Control; EBD, Evans blue dye.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Permeability, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Expressing, Quantitative RT-PCR