Journal: Neural Regeneration Research

Article Title: Enhancing m 6 A modification in the motor cortex facilitates corticospinal tract remodeling after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01477

Figure Lengend Snippet: The antibodies used in the study

Article Snippet: P-P38 , Rabbit , WB: 1:750 , Wanleibio, Shenyang, China , WLP1576 , AB 2922420.

Techniques:

Journal: Neural Regeneration Research

Article Title: Enhancing m 6 A modification in the motor cortex facilitates corticospinal tract remodeling after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01477

Figure Lengend Snippet: METTL14 activates Trib2/MAPK signaling pathway in an m 6 A-dependent manner. (A) Volcano plot of upregulated and downregulated genes in the sham and 7 dpi groups. (B) The top 15 upregulated GO terms in sensorimotor neurons 7 days after SCI. (C) Relative expression of the top 10 upregulated genes in NC- Mettl14 and sh- Mettl14 treated neurons by qPCR ( n = 3 per group). (D) Western blot of TRIB2 in NC- Mettl14 and sh- Mettl14 treated neurons. (E) Quantification of relative protein expression levels in D ( n = 3 per group). (F) Pattern diagram of meRIP-qPCR for Trib2 m 6 A modified site detection. (G) m 6 A level of Trib2 in NC- Mettl14 and sh- Mettl14 treated neurons by meRIP-qPCR ( n = 3 per group). (H) Western blots of TRIB2, p-JNK and p-P38 protein expression in the sham and SCI groups. (I) Quantification of TRIB2, p-JNK and p-P38 protein expression levels in H ( n = 3 per group). (L) Representative images of neurons (NeuN, green, AF488) and TRIB2 expression (red, AF594) in the sham and SCI groups, showing that SCI increased NeuN + TRIB2 + signaling in the cerebral cortex sections. Scale bar: 50 μm. (M) Quantification of the number of TRIB2 + NeuN + cells in L ( n = 4 per group). The data are presented as means ± SD. * P < 0.05, ** P < 0.01 (unpaired t -test (C, E, G, I–K, M)). DAPI: 4′,6-Diamidino2-phenylindole; GO: gene ontology; JNK: Jun N terminal kinase; MeRIP: Methylated RNA immunoprecipitation; m 6 A: N 6 -methyladenosine; Mettl14: methyltransferase-like protein 14; MAPK: mitogen-activated protein kinase; meRIP-qPCR: m 6 A-immunoprecipitation-qPCR; NC: negative control; ns: not significant; p-JNK: phospho-JNK; p-P38: phospho-P38; qPCR: quantitative polymerase chain reaction; SCI: spinal cord injury; Sh: short hairpin; Trib2: tribbles pseudokinase 2.

Article Snippet: P-P38 , Rabbit , WB: 1:750 , Wanleibio, Shenyang, China , WLP1576 , AB 2922420.

Techniques: Expressing, Western Blot, Modification, Methylation, RNA Immunoprecipitation, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Enhancing m 6 A modification in the motor cortex facilitates corticospinal tract remodeling after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01477

Figure Lengend Snippet: METTL14 promotes axon regeneration by activating the Trib2/MAPK pathway. (A) Western blots of TRIB2, p-JNK and p-P38 proteins in NC- Mettl14 , sh- Mettl14 , and sh- Mettl14 +OE- Trib2 treated neurons. (B–D) Quantification of TRIB2, p-JNK and p-P38 protein expression levels in A ( n = 3 per group). (E–L) Relative expression of Ntrk3 , Cntf , Igf1r , NeuroD1 , Nrg1 , Pten , Cers2 , and Ptprs in NC- Mettl14 , sh- Mettl14 , and sh- Mettl14 +OE- Trib2 treated neurons by qPCR ( n = 3 per group). (M) Representative images of immunofluorescence staining of TUJ1 (red, AF594) in NC- Mettl14 , sh- Mettl14 , and sh- Mettl14 +OE- Trib2 treated neurons, showing that overexpression of TRIB2 counteracted the impact of Mettl14 knockdown. Scale bar: 100 μm. (N) Quantification of neurite length in N ( n = 4 per group). (O) Representative immunofluorescence images of the regenerative axons (TUJ1, green, AF488) on the microfluidic cultures, showing that overexpression of TRIB2 counteracted the impact of Mettl14 knockdown. Scale bar: 200 μm. (P) Quantification of total axonal length in G ( n = 4 per group). The data are presented as means ± SD. * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test (B–L, N, P)). Cers2: Ceramide synthase 2; Cntf: ciliary neurotrophic factor; DAPI: 4′,6-diamidino2-phenylindole; Igf1r: insulin like growth factor 1 receptor; JNK: Jun N terminal kinase; MAPK: mitogen-activated protein kinase; Mettl14: methyltransferase-like protein 14; NC: negative control; NeuroD1: neurogenic differentiation 1; Nrg1: neuregulin 1; Ntrk3: neurotrophic receptor tyrosine kinase 3; OE: over-expression; Pten: phosphatase and tensin homolog; Ptprs: protein tyrosine phosphatase receptor type S; Sh: short hairpin; Trib2: tribbles pseudokinase 2.

Article Snippet: P-P38 , Rabbit , WB: 1:750 , Wanleibio, Shenyang, China , WLP1576 , AB 2922420.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Over Expression, Knockdown, Negative Control

Journal: Neural Regeneration Research

Article Title: Hypoxia-preconditioned bone marrow–derived mesenchymal stem cells protect neurons from cardiac arrest–induced pyroptosis

doi: 10.4103/NRR.NRR-D-23-01922

Figure Lengend Snippet: HP-BMSCs suppress OGD-induced neuronal pyroptosis to some extent by inhibiting the MAPK and NF-κB pathways. (A) From left to right: BMSCs at passage 3 (P3), osteogenic differentiation BMSCs, and adipogenic differentiation BMSCs. The cells are typically spindle-shaped. Scale bars: 100 μm. (B) Flow cytometric analysis showing the expression of BMSC surface markers. The cells expressed CD29 and CD90 but did not express CD11b and CD45. (C) Immunofluorescence staining of primary cultured neurons for MAP2 (red, Alexa Fluor 594), whereas nuclei were labeled with DAPI (blue). Scale bars: 75 μm. (D) Western blotting analysis was performed to evaluate the expression of NLRP3, GSDMD-N, ASC, cleaved-caspase-1, NF-κB, JNK, and p38 MAPK in primary neurons from the indicated group ( n = 3). The results of parallel test were shown in Additional Figure 2 . (E–K) Quantitative analysis of NLRP3 (E), GSDMD-N (F), ASC (G), cleaved-caspase-1 (H), NF-κB (I), JNK (J), and p38 MAPK (K) protein expression levels as shown in D. Results are expressed as mean ± SD. * P < 0.05, vs . control group; # P < 0.05, vs . OGD group; & P < 0.05, vs . OGD + N-BMSCs group; $ P < 0.05, vs . OGD + HP-BMSCs group (one-way analysis of variance followed by Tukey’s multiple comparisons test). BMSCs: Bone marrow mesenchymal stem cells; HP-BMSCs: hypoxia preconditioned bone marrow mesenchymal stem cells; N-BMSCs: normal-cultured BMSCs; OGD: oxygen-glucose deprivation.

Article Snippet: Then, the samples were incubated at 4°C overnight with the appropriate primary antibodies: translocase of outer mitochondrial membrane 20 (TOM20, rabbit, 1:5000, Proteintech, Cat# 11802-1-AP, RRID: AB_2207530), fission 1 protein (FIS1, rabbit, 1:1000, Proteintech, Cat# 10956-1-AP, RRID: AB_2102532), cytochrome c oxidase subunit 4 (COX IV, rabbit, 1:5000, Proteintech, Cat# 11242-1-AP, RRID: AB_2085278), brain-derived neurotrophic factor (BDNF, rabbit, 1:1000, Abcam, Cambridge, MA, USA, Cat# ab108319, RRID: AB_10862052), NLRP3 (rabbit, 1:1000, Immunoway, Cat# YT5382, RRID: AB_3095508), apoptosis-associated speck-like protein containing a CARD (ASC, rabbit, 1:1000, Immunoway, Cat# YT0365, RRID: AB_2750947), cleaved-caspase-1 (rabbit, 1:1000, Immunoway, Cat# YC0022, RRID: AB_3095510), gasdermin D N-terminal domain (GSDMD-N, rabbit, Abcam, 1:1000, Cat# ab219800, RRID: AB_2888940), p38 mitogen-activated protein kinase (p38 MAPK, rabbit, 1:1000, Immunoway, Cat# YT3513, RRID: AB_3095512), JNK (rabbit, 1:1000, Immunoway, Cat# YT2440, RRID: AB_2895034), NF-κB p65 (mouse, 1:1000, Immunoway, Cat# YM3111, RRID: AB_3073611), Bcl-2 (mouse, 1:1000, Immunoway, Cat# YM3041, RRID: AB_2814758), bak (mouse, 1:1000, Proteintech, Cat# 29552-1-AP, RRID: AB_2923596), activated-caspase-3 (mouse, 1:1000, Immunoway, Cat# YM3431, RRID: AB_3095515), PFKL (rabbit, 1:1000, Immunoway, Cat# YT3685, RRID: AB_3095516), and β-actin (rabbit, 1:5000, CST, Boston, MA, USA, Cat# 4970, RRID: AB_2223172).

Techniques: Expressing, Immunofluorescence, Staining, Cell Culture, Labeling, Western Blot, Control

Journal: Neural Regeneration Research

Article Title: Hypoxia-preconditioned bone marrow–derived mesenchymal stem cells protect neurons from cardiac arrest–induced pyroptosis

doi: 10.4103/NRR.NRR-D-23-01922

Figure Lengend Snippet: HP-BMSCs suppress OGD-induced neuronal pyroptosis to some extent by inhibiting the MAPK and NF-κB pathways. (A) From left to right: BMSCs at passage 3 (P3), osteogenic differentiation BMSCs, and adipogenic differentiation BMSCs. The cells are typically spindle-shaped. Scale bars: 100 μm. (B) Flow cytometric analysis showing the expression of BMSC surface markers. The cells expressed CD29 and CD90 but did not express CD11b and CD45. (C) Immunofluorescence staining of primary cultured neurons for MAP2 (red, Alexa Fluor 594), whereas nuclei were labeled with DAPI (blue). Scale bars: 75 μm. (D) Western blotting analysis was performed to evaluate the expression of NLRP3, GSDMD-N, ASC, cleaved-caspase-1, NF-κB, JNK, and p38 MAPK in primary neurons from the indicated group ( n = 3). The results of parallel test were shown in Additional Figure 2 . (E–K) Quantitative analysis of NLRP3 (E), GSDMD-N (F), ASC (G), cleaved-caspase-1 (H), NF-κB (I), JNK (J), and p38 MAPK (K) protein expression levels as shown in D. Results are expressed as mean ± SD. * P < 0.05, vs . control group; # P < 0.05, vs . OGD group; & P < 0.05, vs . OGD + N-BMSCs group; $ P < 0.05, vs . OGD + HP-BMSCs group (one-way analysis of variance followed by Tukey’s multiple comparisons test). BMSCs: Bone marrow mesenchymal stem cells; HP-BMSCs: hypoxia preconditioned bone marrow mesenchymal stem cells; N-BMSCs: normal-cultured BMSCs; OGD: oxygen-glucose deprivation.

Article Snippet: Then, the samples were incubated at 4°C overnight with the appropriate primary antibodies: translocase of outer mitochondrial membrane 20 (TOM20, rabbit, 1:5000, Proteintech, Cat# 11802-1-AP, RRID: AB_2207530), fission 1 protein (FIS1, rabbit, 1:1000, Proteintech, Cat# 10956-1-AP, RRID: AB_2102532), cytochrome c oxidase subunit 4 (COX IV, rabbit, 1:5000, Proteintech, Cat# 11242-1-AP, RRID: AB_2085278), brain-derived neurotrophic factor (BDNF, rabbit, 1:1000, Abcam, Cambridge, MA, USA, Cat# ab108319, RRID: AB_10862052), NLRP3 (rabbit, 1:1000, Immunoway, Cat# YT5382, RRID: AB_3095508), apoptosis-associated speck-like protein containing a CARD (ASC, rabbit, 1:1000, Immunoway, Cat# YT0365, RRID: AB_2750947), cleaved-caspase-1 (rabbit, 1:1000, Immunoway, Cat# YC0022, RRID: AB_3095510), gasdermin D N-terminal domain (GSDMD-N, rabbit, Abcam, 1:1000, Cat# ab219800, RRID: AB_2888940), p38 mitogen-activated protein kinase (p38 MAPK, rabbit, 1:1000, Immunoway, Cat# YT3513, RRID: AB_3095512), JNK (rabbit, 1:1000, Immunoway, Cat# YT2440, RRID: AB_2895034), NF-κB p65 (mouse, 1:1000, Immunoway, Cat# YM3111, RRID: AB_3073611), Bcl-2 (mouse, 1:1000, Immunoway, Cat# YM3041, RRID: AB_2814758), bak (mouse, 1:1000, Proteintech, Cat# 29552-1-AP, RRID: AB_2923596), activated-caspase-3 (mouse, 1:1000, Immunoway, Cat# YM3431, RRID: AB_3095515), PFKL (rabbit, 1:1000, Immunoway, Cat# YT3685, RRID: AB_3095516), and β-actin (rabbit, 1:5000, CST, Boston, MA, USA, Cat# 4970, RRID: AB_2223172).

Techniques: Expressing, Immunofluorescence, Staining, Cell Culture, Labeling, Western Blot, Control