Journal: The Journal of Biological Chemistry

Article Title: Fortilin binds and stabilizes MEF2C, activates it through phosphorylation, and drives transcription of the cell structural and survival protein CTNNA3

doi: 10.1016/j.jbc.2026.111417

Figure Lengend Snippet: Fortilin physically binds MEF2C. A, quantitative RT–PCR (qRT–PCR) analysis of THP1 WT-fortilin and THP1 KO-fortilin cells ( n = 3, p < 0.0001, two-sample t test). B – E, MST to characterize the interaction of fortilin with MEF2 family proteins, MEF2A, MEF2B, MEF2C, and MEF2D. K d s were calculated using NanoTemper Analysis software based on the relative F norm values. F – I, in vivo forward co-IP assays. Fortilin was immunoprecipitated from the TCLs of 293T cells using an α-fortilin Ab. Successful IP of fortilin and co-IP of MEF2C and other MEF2 isoforms were assessed by Western blotting using α-fortilin Ab and Abs against MEF2A, MEF2B, MEF2C, and MEF2D, respectively. Total protein was visualized in the SDS-gel containing TCE. J, in vivo reverse co-IP assay. MEF2C was immunoprecipitated from the 293T TCL using an α-MEF2C Ab. Successful IP of MEF2C and co-IP of fortilin were assessed by Western blotting using α-MEF2C and α-fortilin Abs, respectively. Total protein was visualized in an SDS-gel containing TCE. K and L, PLA to evaluate the fortilin–MEF2C interaction. Phorbol 12-myristate 13-acetate-treated THP1 WT-fortilin and THP1 KO-fortilin cells cultured on chamber slides were subjected to a standard PLA assay using rabbit α-fortilin and mouse α-MEF2C Abs. Nuclei were counterstained by DAPI ( blue ). Red puncta indicated that fortilin and MEF2C were located within 30 nm of each other. The scale bar represents 50 μm. K, the PLA interaction index was calculated as the number of PLA puncta per nucleus within a field of view and expressed in AU. Three fields were quantified for each sample, and three independent experiments were performed. Statistical significance was evaluated using a Welch’s two-sample t test ( L ). Ab, antibody; AU, arbitrary unit; DAPI, 4′,6-diamidino-2-phenylindole; α-FLAG, anti-FLAG (DYKDDDDK) antibody (Ab); F norlm , normalized fluorescence value; α-fortilin, rabbit anti-fortilin monoclonal Ab; Fortilin KO, THP1 cells in which the fortilin ( TPT1 ) genes have been deleted by the CRISPR–Cas9 technology (THP1 KO-fortilin ); Fortilin WT, THP1 cells expressing WT fortilin (THP1 WT-fortilin ); α-His 6 , anti-hexahistidine Ab; IB, immunoblot; IgG, normal rabbit immunoglobulin G; INPUT, 10% of TCLs used for IP; IP, immunoprecipitation; α-MEF2C, anti-MEF2C Ab; MST, microscale thermophoresis; PLA, proximity ligation assay; TCE, 2,2,2-trichloroethanol; TCL, total cell lysate.

Article Snippet: We obtained the recombinant human proteins, MEF2A (catalog no.: TP312830; cMYC-FLAG-tagged), MEF2B (catalog no.: TP327214; cMYC-FLAG-tagged), MEF2C (catalog no.: TP320584; cMYC-FLAG-tagged), MEF2D (catalog no.: TP308748; cMYC-FLAG-tagged), and fortilin (catalog no.: TP301664; cMYC-FLAG-tagged) from OriGene and His 6 - NQO2 (catalog no.: ab93933) from Abcam.

Techniques: Quantitative RT-PCR, Software, In Vivo, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, SDS-Gel, Cell Culture, Fluorescence, CRISPR, Expressing, Microscale Thermophoresis, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: Fortilin binds and stabilizes MEF2C, activates it through phosphorylation, and drives transcription of the cell structural and survival protein CTNNA3

doi: 10.1016/j.jbc.2026.111417

Figure Lengend Snippet: Fortilin binds the N-terminal domain of MEF2C. A, MEF2C deletion mutants. Plasmids encoding MEF2C WT and two deletion mutants, MEF2C M1 and MEF2C M2 , lacking residues 1 to 85 and 1 to 270 of MEF2C, respectively, were generated on the pEZ mammalian expression vector. B, in vivo co-IP analysis of MEF2C deletion mutants. TCLs from 293T cells transfected with pEZ-MEF2C WT -FLAG, pEZ-MEF2C M1 -FLAG, or pEZ-MEF2C M2 -FLAG plasmid were subjected to IP in which FLAG-tagged MEF2C proteins were pulled down by α-FLAG Ab and the presence of coimmunoprecipitated fortilin was assessed by Western blotting using α-fortilin Ab. C, sequence alignment of the N-terminal regions of MEF2A, MEF2B, MEF2C, and MEF2D encompassing the MADS-box (residues 3–57) and MEF2 (residues 58–86) domains. Consensus amino acid residues among all four proteins are shown in bold black . Amino acids identical to those in MEF2C are highlighted in yellow . Threonine at position 51, which is uniquely present in MEF2C but absent in MEF2A, MEF2B, and MEF2D, is indicated in bold type . D, HADDOCK scores for fortilin binding to MEF2A, MEF2B, MEF2C, and MEF2D ( n = 4 per the best cluster, one-way ANOVA with Tukey’s multiple comparisons). E, representative docking model showing full-length fortilin ( blue ) in complex with the N-terminal region of MEF2C (red ). Ribbon rendering of the complex is shown with transparent surface representations for both proteins. Amino acid residues critically contributing to the binding interface are indicated for fortilin (Asp 25 , Lys 112 , Glu 120 , and Gln 121 ) in blue and MEF2C (GLN 18 , THR 51 , LYS 53 , LEU 54 , and PHE 55 ) in red . F and G, molecular dynamics simulations of the fortilin–MEF2C complex showing the RMSD time trace ( F ) and RMSD probability distribution ( G ). H, co-IP analysis of the fortilin D25A mutant. THP1 KO-fortilin cells were transiently transfected with pCS mammalian expression vectors encoding HA-tagged WT fortilin (fortilin WT ) or the mutant (fortilin D25A ). TCLs were immunoprecipitated using an anti-HA antibody, and coimmunoprecipitated MEF2C was detected by Western blotting. AU, arbitrary unit; FLAG, an epitope-tag consisting of DYKDDDDK; fortilin WT -HA, pCS-fortilin WT -3×HA plasmid that expresses the WT MEF2C fused to the three hemagglutinin (HA) epitope tag repeats at its C-terminal end; fortilin D25A -HA, pCS-fortilin D25A -3×HA; IP, immunoprecipitation; M1, pEZ-MEF2C M1 -FLAG (amino acids 86–463); M2, pEZ-MEF2C M1 -FLAG (amino acids 271–463); MADS-box, MCM1-Agamous-Deficiens-Serum Response Factor box; MEF2, myocyte enhancer factor-2; MEF2C WT , WT MEF2C (1–463 amino acids); MEF2C M1 , a deletion mutant of MEF2C lacking the N-terminal MADS-box and MEF2 domains and containing amino acids 86 to 463 of WT MEF2C; MEF2C M2 , a deletion mutant containing amino acids 271 to 463 of WT MEF2C; α-MEF2C, anti-MEF2C antibody; α-HA, anti-HA-tag antibody; TCL, total cell lysate; THP1 WT-fortilin , THP1 cells expressing WT fortilin; THP1 KO-fortilin , THP1 cells in which the fortilin genes have been deleted by the CRISPR–Cas9 technology; WT, pEZ-MEF2C WT -FLAG plasmid that expresses the WT MEF2C (amino acids 1–463) fused to the FLAG epitope tag at its C-terminal region.

Article Snippet: We obtained the recombinant human proteins, MEF2A (catalog no.: TP312830; cMYC-FLAG-tagged), MEF2B (catalog no.: TP327214; cMYC-FLAG-tagged), MEF2C (catalog no.: TP320584; cMYC-FLAG-tagged), MEF2D (catalog no.: TP308748; cMYC-FLAG-tagged), and fortilin (catalog no.: TP301664; cMYC-FLAG-tagged) from OriGene and His 6 - NQO2 (catalog no.: ab93933) from Abcam.

Techniques: Generated, Expressing, Plasmid Preparation, In Vivo, Co-Immunoprecipitation Assay, Transfection, Western Blot, Sequencing, Binding Assay, Mutagenesis, Immunoprecipitation, CRISPR, FLAG-tag

Journal: The Journal of Biological Chemistry

Article Title: Fortilin binds and stabilizes MEF2C, activates it through phosphorylation, and drives transcription of the cell structural and survival protein CTNNA3

doi: 10.1016/j.jbc.2026.111417

Figure Lengend Snippet: Fortilin binds the transcription factor MEF2C, protects it against proteasomal degradation, enhances its phosphorylation, and transcriptionally activates the CTNNA3 promoter. Under normal conditions (Fortilin [+], left panel ), fortilin binds MEF2C and protects it from proteasomal degradation ① . In addition, fortilin facilitates the phosphorylation of MEF2C ② , thereby enhancing its DNA binding capacity and promoting transcriptional activation of the CTNNA3 gene ③ . In contrast, under fortilin-deficient conditions (Fortilin [–], right panel ), MEF2C undergoes increased proteasomal degradation and becomes less phosphorylated at Ser 59 , which impairs its ability to bind the CTNNA3 promoter and transcriptionally activate the gene, ultimately leading to cellular dysfunction. FT, fortilin; P, phosphorylation.

Article Snippet: We obtained the recombinant human proteins, MEF2A (catalog no.: TP312830; cMYC-FLAG-tagged), MEF2B (catalog no.: TP327214; cMYC-FLAG-tagged), MEF2C (catalog no.: TP320584; cMYC-FLAG-tagged), MEF2D (catalog no.: TP308748; cMYC-FLAG-tagged), and fortilin (catalog no.: TP301664; cMYC-FLAG-tagged) from OriGene and His 6 - NQO2 (catalog no.: ab93933) from Abcam.

Techniques: Phospho-proteomics, Binding Assay, Activation Assay

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A-E) Confirmation of Lenti-Mef2D overexpression line using mRNA and protein. Top band and bottom band of samples quantified. F-H) Confirmation of Lenti-ShMef2D knockdown line using mRNA and protein. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques: Over Expression, Knockdown

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: Oxidative phosphorylation was measured in both Mef2D overexpression and knockdown lines. A, F) Glutamate and Malate (G, M) used to stimulate Complex I. B, G) Succinate to stimulate Complex II. C, H) ADP is used to stimulate Complex V. D, I) Rotenone is used to inhibit Complex I activity to allow data to be gathered for Complex II linked oxidative phosphorylation. E, J) FCCP is a proton uncoupler to allow for measurement of the maximal respiration of the cells. K-O) No treatment was compared with Lenti-ShCtrl across the steps to confirm no change with lentiviral induction. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques: Phospho-proteomics, Over Expression, Knockdown, Activity Assay

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A, B) mRNA measurement of Ndufv3 in Lenti-Mef2D and Lenti-ShMef2D. C-E) Protein and mRNA measurement of mtND6 in Lenti-Mef2D. F-J) Protein measurement of electron transport chain components Complex I, II, III, and V. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques:

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A-F) mRNA measurement of dehydrogenases in TCA and ETC of Lenti-Mef2D. G-L) mRNA measurement of dehydrogenases in TCA and ETC of Lenti-ShMef2D. M,R) mRNA measurement of Insulin in Lenti-Mef2D and Lenti-ShMef2D respectively. N-P) mRNA and protein measurement of GLUT2 in Lenti-Mef2D. T, U) Protein measurement of GLUT2 in Lenti-ShMef2D. Q, S) mRNA measurement of Nr4a1 in Lenti-Mef2D and Lenti-ShMef2D respectively. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques:

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A) Glucose stimulated insulin secretion in Lenti-Mef2D line is inhibited. B) Insulin content in Lenti-Mef2D. C) Glucose stimulated insulin secretion in Lenti-ShMef2D is enhanced. D) Insulin content in Lenti-ShMef2D is enhanced. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques:

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A) Alamar blue measurement of cell viability in Lenti-Mef2D treated with Etoposide, Camptothecin, Thapsigargin. B) Alamar blue measurement of cell viability in Lenti-ShMef2D treated with Etoposide, Camptothecin, Thapsigargin. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques:

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A) Mef2D overexpression will reduce the expression of the ETC components mtND6, Ndufv3, and SDHB, reducing mitochondrial respiration. The TCA dehydrogenase Ogdh will add to the reduced mitochondrial respiration. Reduced mitochondrial respiration, along with reduced GLUT2 expression, will lead to reduced GSIS. Reduced insulin mRNA will add to the reduced GSIS. Mitochondrial respiration is also leading to reduced cell viability. B) Mef2D knockdown leads to enhanced mitochondrial respiration and increased insulin content, leading to increased GSIS. The increased mitochondrial respiration, along with increased Nr4a1, will also lead to increased cell viability.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques: Over Expression, Expressing, Knockdown

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A-E) Confirmation of Lenti-Mef2D overexpression line using mRNA and protein. Top band and bottom band of samples quantified. F-H) Confirmation of Lenti-ShMef2D knockdown line using mRNA and protein. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques: Over Expression, Knockdown

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: Oxidative phosphorylation was measured in both Mef2D overexpression and knockdown lines. A, F) Glutamate and Malate (G, M) used to stimulate Complex I. B, G) Succinate to stimulate Complex II. C, H) ADP is used to stimulate Complex V. D, I) Rotenone is used to inhibit Complex I activity to allow data to be gathered for Complex II linked oxidative phosphorylation. E, J) FCCP is a proton uncoupler to allow for measurement of the maximal respiration of the cells. K-O) No treatment was compared with Lenti-ShCtrl across the steps to confirm no change with lentiviral induction. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques: Phospho-proteomics, Over Expression, Knockdown, Activity Assay

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A, B) mRNA measurement of Ndufv3 in Lenti-Mef2D and Lenti-ShMef2D. C-E) Protein and mRNA measurement of mtND6 in Lenti-Mef2D. F-J) Protein measurement of electron transport chain components Complex I, II, III, and V. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques:

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A-F) mRNA measurement of dehydrogenases in TCA and ETC of Lenti-Mef2D. G-L) mRNA measurement of dehydrogenases in TCA and ETC of Lenti-ShMef2D. M,R) mRNA measurement of Insulin in Lenti-Mef2D and Lenti-ShMef2D respectively. N-P) mRNA and protein measurement of GLUT2 in Lenti-Mef2D. T, U) Protein measurement of GLUT2 in Lenti-ShMef2D. Q, S) mRNA measurement of Nr4a1 in Lenti-Mef2D and Lenti-ShMef2D respectively. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques:

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A) Glucose stimulated insulin secretion in Lenti-Mef2D line is inhibited. B) Insulin content in Lenti-Mef2D. C) Glucose stimulated insulin secretion in Lenti-ShMef2D is enhanced. D) Insulin content in Lenti-ShMef2D is enhanced. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques:

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A) Alamar blue measurement of cell viability in Lenti-Mef2D treated with Etoposide, Camptothecin, Thapsigargin. B) Alamar blue measurement of cell viability in Lenti-ShMef2D treated with Etoposide, Camptothecin, Thapsigargin. p-values *<0.05, **<0.01, ***<0.001, or ****<0.0001.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques:

Journal: bioRxiv

Article Title: MEF2D impairs mitochondrial respiration, glucose-stimulated insulin secretion, and survival in INS-1 β-cells

doi: 10.64898/2026.03.07.709963

Figure Lengend Snippet: A) Mef2D overexpression will reduce the expression of the ETC components mtND6, Ndufv3, and SDHB, reducing mitochondrial respiration. The TCA dehydrogenase Ogdh will add to the reduced mitochondrial respiration. Reduced mitochondrial respiration, along with reduced GLUT2 expression, will lead to reduced GSIS. Reduced insulin mRNA will add to the reduced GSIS. Mitochondrial respiration is also leading to reduced cell viability. B) Mef2D knockdown leads to enhanced mitochondrial respiration and increased insulin content, leading to increased GSIS. The increased mitochondrial respiration, along with increased Nr4a1, will also lead to increased cell viability.

Article Snippet: Rat shRNA constructs against Mef2D (5’-TTTCCGTGGCAACACCAAGTTTACTCAGC-3’) and a control 29-mer scrambled shRNA cassette (termed shCTRL) were cloned into the pGFP-C-shLenti plasmid, as were Lentivirus rat Mef2D overexpression particles and control GFP overexpression particles were produced and acquired from OriGene.

Techniques: Over Expression, Expressing, Knockdown

Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) RT-PCR analysis of the mutually exclusive α-exons in Mef2d transcript using total RNA from the indicated fetal hind limb muscle and tissues from adult wild-type mice. ( B ) RT-PCR analysis of Mef2d α exons using soleus RNA from line 2. The numbers indicate PSI; data are mean ± SD; n = 3. The numbers indicate the percent spliced in (PSI). Bolded numbers are significant by Student T test. ( C ) RT-PCR analysis of the alternative exons in Mef2c and Mef2a transcripts using total RNA from gastrocnemius muscle from line 1. Data are mean ± SD; n = 3. PSI for exon is indicated and was not significantly different between the genotypes by Student’s t test. ( D ) Western blot showing MEF2D protein levels in TA (upper left) and Soleus muscles (upper right) from line 2. The bottom panels show Coomassie stained blots, from the upper panels showing total protein loaded. The numbers are mean ± SD; n = 3. Student’s t test found no differences in genotypes. ( E ) RT-qPCR showing relative mRNA levels Mef2d , Mef2a , and Mef2c relative to Rpl30 using total RNA in the indicated muscle groups from line 2 mice. Data are mean ± SEM; n = 4. ** P = 0.0088 (multiple Student’s t test, unpaired). .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Muscles, Staining, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) RT-PCR analysis of the mutually exclusive α-exons and alternative β exon in Mef2d transcript using total RNA from the indicated muscle groups from Wt and Mef2dα2 Eko mice from line 1. The numbers indicate the percent spliced in (PSI) for indicated exon. Data are mean ± SD; n = 3. Bold numbers indicate significance by Student t test. ( B ) Western blot showing MEF2D protein levels in TA (upper left) and Soleus muscles (upper right) from line 1. Bottom panels show Coomassie stained blots from the upper panels showing total protein loaded. The numbers indicate the relative protein level normalized to total protein by Coomassie stained blots. Data are mean ± SD; n = 3. ( C ) RT-qPCR showing mRNA levels Mef2d , Mef2a , and Mef2c relative to Rpl30 using total RNA in the indicated muscle groups from line 1 mice. Data are mean ± SEM; n = 3. No significant changes were found between the genotypes by multiple t test in ( B , C ). .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Muscles, Staining, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) RNA-sequencing data using poly-A selected RNA from WT and MEF2Dα2 Eko quadriceps muscles were aligned to mm10 UCSC browser showing Mef2d α exons region. ( B ) Graph showing number of genes that were down or upregulated in MEF2Dα2 Eko muscles in comparison to age and sex-matched WT muscles (FDR < 0.05). ( C ) RT-qPCR showing relative expression of Bdh1, Oxct1, and Acat1 transcripts normalized to Hprt transcript levels in WT and MEF2Dα2 Eko soleus muscles from line 1. Data are mean ± SEM; n ≥ 6. ** P < 0.01, *** P < 0.001 (Student’s t test). The exact P value is indicated above the bar graphs. ( D ) Western blot showing BDH1 level in soleus muscles of indicated mice from line 1. The panel on the right show BDH1, OXCT1, and ACAT1 level when normalized to total protein loaded as estimated by Coomassie staining of the same blot. Data are mean ± SEM, n = 3, * P < 0.016, ** P < 0.0042, **** P = 0. 000082 (Student’s t test). .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: RNA Sequencing, Muscles, Comparison, Quantitative RT-PCR, Expressing, Western Blot, Staining

Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) β-hydroxybutyrate (BHB) tolerance test in untrained 28-week-old male sedentary mice. Graphs displays mean ± SEM, with n ≥ 9. * P < 0.05 ( P = 0.02525 at 45 min, P = 0.021921 at 60 min), ** P < 0.01 ( P = 0.002777 at 90 min) by multiple t test between genotypes at different time-points. ( B ) The indicated age-matched male mice were run for 55 min at 18 m/min, and BHB was measured immediately after running (0 h), and 1–3 h post-run. The pre-run BHB values are from the same mice a day before the experiment. Data are mean ± SD, n = 10, n.s. not significant; P > 0.05, ** P < 0.01 ( P = 0.00121 at 0 h), * P < 0.05 ( P = 0.0485 at 2-h post-exercise), *** P < 0.001 (0.000233088 at 3-h post-exercise) by multi p le t test between genotypes at different time-points. ( C ) The indicated age-matched male mice were fed control or ketogenic diet for 2-weeks and BHB was measured without fasting. Data are mean ± SD; n ≥ 8. The exact P value as calculated by one-way ANOVA is indicated above the bar graphs. ( D ) Comparison between state 3 JO 2 (ADP 200 µM) for isolated mitochondria from WT and MEF2Dα2 Eko skeletal muscles in presence of alpha-ketoglutarate (AKG) and AKG+Acetoacetate (AcAc). The representative time courses for these experiments are shown in Fig. . Data are mean ± SD; n = 5. The exact P value as calculated by one-way ANOVA is indicated above the bar graphs. ( E ) ChIP quantitative PCR for indicated genes using MEF2D and Serotype control (IgG) are shown (left), representative DNA product visualized on a 5% acrylamide gel (right). Data are mean ± SD; n = 4. For Bdh1, actual P value is indicated; for others, * P < 0.05, ** P < 0.01 (paired Student’s t test). The exact P value is indicated above the bar graphs. .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: Control, Comparison, Isolation, Muscles, Real-time Polymerase Chain Reaction, Acrylamide Gel Assay

Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) RT-qPCR showing relative expression of indicated transcripts normalized to Hprt transcript levels using total RNA from WT and MEF2Dα2 Eko mice livers. Data are mean ± SEM, n = 5, Student’s t test found no differences in genotypes. ( B ) Representative time-courses of isolated mitochondrial respiration for WT and MEF2Dα2 Eko mice transitioning from state 1 to state 4 respiration under AKG± AcAc. The respiratory rates are expressed as nmol/min/mg mitochondrial protein. The transitions from state 1 to state 4 respiration were monitored by first adding isolated mitochondria (0.05 mg/mL) to the respiration buffer at t = 0 min leading to state 1. At t = 2 min, substrates were added to energize the mitochondria, which led to state 2 respiration. This was followed by sequential additions of incremental ADP concentrations (100 and 200 µM). AKG: Alpha-ketoglutarate and AcAc: acetoacetate. ( C ) MEF2D ChIP data from Gönczi et al viewed on IGV viewer (version 2.19.4) for Igfn1, Bdh1, Oxct1, and Acat1 gene locus. The bi-directional arrowed line shows the region where we designed our primers for our analyses. .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: Quantitative RT-PCR, Expressing, Isolation