Journal: International Journal of Surgery (London, England)

Article Title: Inhibition of Mettl3-mediated m6A RNA modification of HMGCS1 protects retinal ganglion cells from glutamate excitotoxicity-induced ferroptosis in a rat model of glaucoma

doi: 10.1097/JS9.0000000000003213

Figure Lengend Snippet: m6A RNA methylation levels and methyltransferase expression changes in glutamate excitotoxicity-induced glaucoma model of rat. (A) A schematic model for the mechanism of NMDA-induced retinal excitotoxic in RGCs. (B) Schematic diagram of constructing NMDA-induced glaucoma model of rat. Qualitative observation (C) and quantitative analysis (D) of the NMDA-induced RGC injury in rat retina (n = 5). Three days after intravitreal injection of NMDA, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGCs exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 100 μm. (E) mRNA level of Mettl3 and FTO in retina of NMDA-induced glaucoma model of rat. (n = 3) (F) The RNA methylation level of poly(A) + RNAs isolated from total RNA of the retina of NMDA-induced glaucoma model of rat was indicated by m6A dot blot. Corresponding RNAs were loaded equally by a 2-fold serial dilution with 400 ng, 200 ng and 100 ng. Methylene blue staining served as a loading control. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated.

Article Snippet: According to the manufacturer’s protocol, DynabeadsTM Antibody Coupling Kit (14311D; Invitrogen, California, United States) was pre-incubated with the m6A antibody (#202003; Synaptic Systems, Germany) to generate m6A-immunomagnetic beads.

Techniques: Methylation, Expressing, Injection, Labeling, Fluorescence, Microscopy, Isolation, Dot Blot, Serial Dilution, Staining, Control, Comparison

Journal: International Journal of Surgery (London, England)

Article Title: Inhibition of Mettl3-mediated m6A RNA modification of HMGCS1 protects retinal ganglion cells from glutamate excitotoxicity-induced ferroptosis in a rat model of glaucoma

doi: 10.1097/JS9.0000000000003213

Figure Lengend Snippet: m6A RNA methylation. (A) Distribution of peak on gene functional elements. (B) Peak density in different region. (C) Enrichment for known m6A consensus motif RRACH. (D) Volcano plot of differentially expressed genes between NMDA and control groups. (E) The heat map shows NMDA-induced top 20 genes. (F) m6A-seq and RNA-seq analysis of differentially expressed genes between NMDA and control group.

Article Snippet: According to the manufacturer’s protocol, DynabeadsTM Antibody Coupling Kit (14311D; Invitrogen, California, United States) was pre-incubated with the m6A antibody (#202003; Synaptic Systems, Germany) to generate m6A-immunomagnetic beads.

Techniques: Methylation, Functional Assay, Control, RNA Sequencing

Journal: International Journal of Surgery (London, England)

Article Title: Inhibition of Mettl3-mediated m6A RNA modification of HMGCS1 protects retinal ganglion cells from glutamate excitotoxicity-induced ferroptosis in a rat model of glaucoma

doi: 10.1097/JS9.0000000000003213

Figure Lengend Snippet: HMGCS1 inhibits ferroptosis in R28 cells in an m6A-dependent manner. (A) Effect of STM2457 (25 μM) and FB23 (50 μM) on the cell viability of R28 cells treated with 5, 10, 15, 20, 25 mM glutamate for 24 hours (n = 3). (B, C) Effect of STM2457 (25 μM) and FB23 (50 μM) on the cell viability of R28 cells (n = 3). (D) MeRIP-qPCR analysis of HMGCS1 after treated with glutamate (10 mM) and STM2457 (25 μM) for 24 hours in R28 cells. (E) Mettl3 and HMGCS1 protein expression in R28 cells treated with glutamate (10 mM) and STM2457 (25 μM) for 24 hours (n = 3). (F) Confocal microscopy was used to detect the intracellular HMGCS1 protein levels. The protein was labeled with red fluorescence, the cytoskeleton was labeled with green fluorescence, and the nucleus was stained with DAPI, showing blue fluorescence. (G) mRNA level of HMGCS1 in R28 cells treated with glutamate (10 mM) and STM2457 (25 μM) for 24 hours (n = 3). (H) Quantification analysis of HMGCS1 protein expression in R28 cells (n = 3). (I) Quantification analysis of Mettl3 protein expression in R28 cells (n = 3). (J) Quantification analysis of HMGCS1 protein mean fluorescence intensity in R28 cells (n = 5). (K) HMGCS1, SLC7A11 and GPX-4 protein expression in R28 cells with HMGCS1 knockdown or overexpress (n = 3). Quantification analysis of HMGCS1 (L), SLC7A11 (M) and GPX4 (N) protein expression in R28 cells (n = 3). (O) Confocal microscopy was used to detect the intracellular HMGCS1 protein levels. (P) Quantification analysis of HMGCS1 protein mean fluorescence intensity in R28 cells (n = 5). (Q) Confocal microscopy was used to detect the intracellular SLC7A11 protein levels. (R) Quantification analysis of SLC7A11 protein mean fluorescence intensity in R28 cells (n = 5). (S) Confocal microscopy was used to detect the intracellular GPX4 protein levels. (T) Quantification analysis of GPX4 protein mean fluorescence intensity in R28 cells (n = 5). (U) PI (red) and Calcein AM (green) staining of R28 cells treated with glutamate, STM2457, si-HMGCS1 or oe-HMGCS1 as indicated for 24 hours. Scale bar: 50 μm. (V) Quantification of the results shown in U (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001, ns: P > 0.05 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated.

Article Snippet: According to the manufacturer’s protocol, DynabeadsTM Antibody Coupling Kit (14311D; Invitrogen, California, United States) was pre-incubated with the m6A antibody (#202003; Synaptic Systems, Germany) to generate m6A-immunomagnetic beads.

Techniques: Expressing, Confocal Microscopy, Labeling, Fluorescence, Staining, Knockdown, Comparison

Journal: International Journal of Surgery (London, England)

Article Title: Inhibition of Mettl3-mediated m6A RNA modification of HMGCS1 protects retinal ganglion cells from glutamate excitotoxicity-induced ferroptosis in a rat model of glaucoma

doi: 10.1097/JS9.0000000000003213

Figure Lengend Snippet: YTHDF2 specifically recognizes the m6A-methylated sites on HMGCS1 mRNA, mediating the degradation of HMGCS1 RNA. (A–C) YTHDF2 mRNA and protein expression in R28 cells with YTHDF2 knockdown. (D, E) HMGCS1 mRNA and protein expression in R28 cells with YTHDF2 knockdown. Confocal microscopy was used to detect the intracellular YTHDF2 (F) and HMGCS1 (G) protein levels. The protein was labeled with red fluorescence, the cytoskeleton was labeled with green fluorescence, and the nucleus was stained with DAPI, showing blue fluorescence. (H, I) Quantification analysis of YTHDF2 and HMGCS1 protein mean fluorescence intensity in R28 cells (n = 5). (J) HMGCS1 mRNA decay assay using actinomycin D treatment in R28 cells transfected with si-YTHDF2 (n = 3). (K) PI (red) and Calcein-AM (green) staining of R28 cells treated with glutamate, STM2457 and si-HMGCS1 as indicated for 24 hours. Scale bar: 50 μm. (L) Quantification of the results shown in K (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated.

Article Snippet: According to the manufacturer’s protocol, DynabeadsTM Antibody Coupling Kit (14311D; Invitrogen, California, United States) was pre-incubated with the m6A antibody (#202003; Synaptic Systems, Germany) to generate m6A-immunomagnetic beads.

Techniques: Methylation, Expressing, Knockdown, Confocal Microscopy, Labeling, Fluorescence, Staining, Mrna Decay Assay, Transfection, Comparison