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–MEF2C interaction mitigates the ubiquitination and proteasomal degradation of MEF2C. A – D, time-course analysis of MEF2C degradation in the presence or the absence of carfilzomib using the JESS capillary-based Western blot system. THP-1 cells were transiently transfected with pEZ-MEF2C WT -FLAG, incubated for 48 h, treated with CHX in the presence or the absence of carfilzomib (defined as time 0), and harvested at the indicated time points. TCLs were analyzed by IB with α-FLAG and α-fortilin Abs to assess MEF2C WT -FLAG and fortilin expression, respectively. Total protein loading was visualized using the JESS total protein detection module ( A ). Quantitative analysis of MEF2C expression and degradation in THP1 WT-fortilin and THP1 KO-fortilin cells treated with and without carfilzomib. Four experimental groups were analyzed—THP1 WT-fortilin ± carfilzomib and THP1 KO-fortilin ± carfilzomib. MEF2C expression indices (AU) were calculated at each time point using JESS Compass software by dividing the area under the MEF2C peak by the total protein signal within the same capillary (“in-capillary normalization”). Absolute MEF2C expression levels at time 0 were first compared between THP1 WT-fortilin and THP1 KO-fortilin cells in the absence ( B ) and presence ( D ) of carfilzomib. Data are presented as means ± standard deviation with p values indicated ( n = 3, two-sample t test). MEF2C degradation kinetics were then assessed by normalizing MEF2C expression indices at each time point to the corresponding time 0 value within each group and comparing degradation profiles between THP1 WT-fortilin and THP1 KO-fortilin cells in the absence and presence of carfilzomib ( n = 3, two-sample t test) ( C ). E and F, MEF2C ubiquitination assay. MEF2C was immunoprecipitated from the TCLs of THP1 WT-fortilin and THP1 KO-fortilin cells using an anti-MEF2C Ab, and ubiquitinated MEF2C was detected by IB with an anti-ubiquitin Ab ( E ). MEF2C ubiquitination indices were calculated by normalizing the total signal intensity of ubiquitinated MEF2C to the signal intensity of unmodified MEF2C in the TCLs ( n = 3, two-sample t test) ( F ). G and H, effect of fortilin binding to MEF2C on MEF2C degradation. THP1 KO-fortilin cells were transfected with plasmids encoding fortilin WT -HA or fortilin D25A -HA (an MEF2C-binding–deficient mutant), treated with CHX, and incubated for 12 h in the presence or the absence of carfilzomib. TCLs were analyzed by Western blotting using anti-MEF2C and anti-HA Abs ( G ). MEF2C expression indices were calculated by normalizing the area under the curve of the MEF2C peak to total protein using Compass software (ProteinSimple, n = 3, ANOVA with Tukey’s multiple comparisons) ( H ). AU, arbitrary unit; carfilzomib, proteasome inhibitor; CHX, cycloheximide; α-FLAG, anti-FLAG antibody (Ab); α-fortilin, anti-fortilin Ab; fortilin D25A -HA, a HA-tagged MEF2C-binding–deficient fortilin mutant in which the 25th aspartic acid ( D ) is mutated to alanine ( A ); fortilin WT -HA, HA-tagged WT fortilin; IB, immunoblot; α-MEF2C, anti-MEF2C Ab; MG132, carbobenzoxy-L-leucyl-L-leucyl-L-leucine (proteasome inhibitor); TCL, total cell lysate; THP1 KO-fortilin , THP1 cells in which the fortilin gene was deleted by CRISPR–Cas9 (KO); THP1 WT-fortilin , THP1 cells expressing WT fortilin (WT); α-UB, anti-ubiquitin Ab; UB(n)-MEF2C, ubiquitinated MEF2C.

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: Ubiquitin Proteomics, Western Blot, Transfection, Incubation, Expressing, Software, Standard Deviation, Immunoprecipitation, Binding Assay, Mutagenesis, CRISPR

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 promotes the phosphorylation of MEF2C at Ser59 in the N-terminal region. A, phosphorylation status of WT MEF2C in THP1 WT-fortilin and THP1 KO-fortilin cells. THP1 WT-fortilin and THP1 KO-fortilin cells were transiently transfected with the pEZ-MEF2C WT -FLAG plasmid (mock transfection served as a control). MEF2C WT -FLAG was immunoprecipitated from the TCLs using an α-FLAG Ab and subjected to SDS-PAGE. Phosphorylated MEF2C WT protein was visualized by Pro-Q Diamond staining ( top panel ), and total MEF2C WT protein was visualized by SYPRO Ruby staining ( bottom panel ). B, comparison of the phosphorylation status of MEF2C WT and MEF2C M1 in the presence (THP1 WT-fortilin ) and absence (THP1 KO-fortilin ) of fortilin. THP1 WT-fortilin and THP1 KO-fortilin cells were transiently transfected with either pEZ-MEF2C WT -FLAG or pEZ-MEF2C M1 -FLAC plasmids. FLAG-tagged MEF2C WT and MEF2C KO proteins were then immunoprecipitated from the TCLs using an α-FLAG Ab and subjected to SDS-PAGE. Phosphorylated MEF2C proteins were visualized by Pro-Q Diamond staining ( top panel ), and total MEF2C proteins were visualized by SYPRO Ruby staining ( bottom panel ). C – G, evaluation of MEF2C phosphorylation at Ser 59 in the presence and absence of fortilin. Endogenous MEF2C was immunoprecipitated from the TCLs of THP1 WT-fortilin (WT) and THP1 KO-fortilin (KO) cells using anti-MEF2C antibody. Total immunoprecipitated MEF2C and Ser 59 -phosphorylated MEF2C were analyzed using the JESS Western blot system ( C – E ). MEF2C phosphorylation indices were calculated from three independent experiments as the ratio of the area under the curve of phosphorpho-MEF2C signal to that of the total MEF2C signal and expressed in AU. A two-sample t test was used to compare the means of the WT and KO phosphorylation indices ( F and G ). H – J, evaluation of the role of fortilin–MEF2C binding in maintaining MEF2C phosphorylation at Ser 59 . THP1 KO-fortilin cells were transiently transfected with plasmids encoding 3xHA-tagged fortilin WT or fortilin D25A or with nothing. TCLs were analyzed using the JESS Western blot system with α-MEF2C, α-HA, and α-phospho-Ser 59 -MEF2C Abs ( H , I ). The MEF2C phosphorylation index was calculated as the ratio of Ser 59 -phosphorylated MEF2C to total MEF2C ( n = 3; one-way ANOVA with Tukey’s multiple-comparisons test) ( J ). AU, arbitrary unit; α-FLAG, anti-FLAG antibody (Ab); fortilin D25A -HA, pCS-fortilin D25A -3×HA plasmid that expresses the MEF2C-binding–deficient fortilin D25A mutant fused to the three hemagglutinin (HA) epitope tag repeats at its C-terminal end; fortilin WT -HA, pCS-fortilin WT -3×HA plasmid that expresses the WT fortilin fused to the three HA epitope tag repeats at its C-terminal end; α-HA, anti-HA Ab; IB, immunoblot; IP, immunoprecipitation; INPUT, 10% of TCLs used for IP; JESS, an automated capillary-based quantitative Western blot system; KO, THP1 KO-fortilin ; M1, pEZ-MEF2C M1 -FLAG; α-MEF2C, anti-MEF2C Ab; pEZ-MEF2C WT -FLAG, a mammalian expression plasmid containing the construct for FLAG-tagged MEF2C; Pro-Q Diamond, staining for phosphorylated protein; α-Ser 59 , anti-MEF2C-Ser 59 Ab; SYPRO Ruby, staining for total protein; TCL, total cell lysate; WT, THP1 WT-fortilin .

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, Transfection, Plasmid Preparation, Control, Immunoprecipitation, SDS Page, Staining, Comparison, Western Blot, Binding Assay, Mutagenesis, Expressing, Construct

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 enhances MEF2C-mediated transcriptional activation of the CTNNA3 promoter. A and B, PLA. Interaction between MEF2C and dsDNA evaluated by PLA. PLA was performed using rabbit α-MEF2C and mouse α-dsDNA Abs in THP1 WT-fortilin (WT, top panels ) and THP1 KO-fortilin (KO, bottom panels ) cells, with nuclei counterstained using DAPI ( blue ). Red puncta indicate that MEF2C and dsDNA are located within 30 nm of each other, signifying a specific interaction. The scale bar represents 10 μm. White squares indicate magnified regions shown below ( A ). The PLA interaction index was calculated as the number of PLA puncta per nucleus within a field of view and is expressed in arbitrary units (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 ( B ). C, structure of the human CTNNA3 gene ( top panel ) and pGL-P CTNNA3 -Luc reporter vector ( bottom panels ). A high-probability MEF2C binding site was identified within the 3266-nucleotide CTNNA3 promoter region using the JASPAR platform. This 3266-nucleotide CTNNA3 promoter region that contains a top-scoring, predicted MEF2C binding motif (ATAAAAATACA) located from −976 to −966 relative to the transcription start site (TSS) was cloned upstream of the firefly luciferase reporter gene in the pGL vector to generate pGL-P CTNNA3-Luc vector (total size = 8230 base pairs). D, dual luciferase reporter assay to assess the impact of fortilin on CTNNA3 promoter activation. 293T cells were sequentially transfected with siRNA targeting fortilin (siRNA fortilin ) or control siRNA (siRNA control ), followed by cotransfection with pRL-null vector ( Renilla luciferase, normalization control) and either pGL-P Control-Luc or pGL-P CTNNA3 -Luc reporter plasmids. Luciferase activity was measured 24 h post-transfection, and the RAI was calculated as the ratio of firefly to Renilla luciferase activity. Data are expressed as means ± standard deviation ( n = 3 biological replicates) and were analyzed using two-way ANOVA followed by Tukey–Kramer multiple comparisons. E and F, dual luciferase reporter assay to determine whether MEF2C is required for fortilin-mediated activation of the CTNNA3 promoter. 293T cells were transfected with one of the following siRNA combinations: (i) siRNA MEF2C + siRNA control , (ii) siRNA MEF2C + siRNA fortilin , (iii) siRNA control , or (iv) siRNA fortilin . Luciferase activity was measured 24 h post-transfection, and the RAI was calculated. Data are presented as means ± standard deviation ( n = 4) and were analyzed using two-way ANOVA and Tukey’s multiple comparisons ( E ). Subsequently, cell lysates were subjected to Western blotting using α-MEF2C and α-fortilin Abs to confirm siRNA-mediated knockdown of target genes ( F ). G and H, TATA-binding protein (TBP) and RNA polymerase II (Pol II) chromatin immunoprecipitation (ChIP) of CTNNA3. ChIP assays were performed in THP1 WT-fortilin and THP1 KO-fortilin cells by crosslinking chromatin with formaldehyde, followed by immunoprecipitation of chromatin from the TCLs using an anti-TBP ( G ) or anti-Pol II ( H ) antibody coupled to Protein A/G beads. Normal IgG was used as a negative control. DNA from the immunoprecipitated chromatin was purified and analyzed by quantitative PCR (qPCR) using primers flanking the transcription start sites (TSSs) of CTNNA3 , CD68 (positive control), and V WF (negative control). TBP ( G ) and Pol II ( H ) occupancy indices for CTNNA3, CD68, and vWF were expressed as fold enrichment values (AU) relative to IgG ( n = 4; Student's t test for TBP ( G ) and Welch’s t tests for Pol II ( H ). I and J, CTNNA3 expression in THP1 KO-fortilin cells overexpressing WT (fortilin WT ) and an MEF2C-binding–deficient (fortilin D25A ) fortilin. THP1 KO-fortilin cells were transiently transfected with plasmids encoding HA-tagged fortilin WT or fortilin D25A . Untransfected THP1 KO-fortilin cells were used as controls. TCLs were analyzed using the JESS automated Western blot system with anti-HA and anti-CTNNA3 antibodies ( I ). The CTNNA3 expression index was calculated by normalizing the area under the CTNNA3 peak to the total protein signal and expressed as arbitrary units (AU) ( n = 3; one-way ANOVA with Tukey’s multiple comparisons) ( J ). AU, arbitrary unit; α-CTNNA3, anti-CTNNA3 antibody; ChIP–qPCR, chromatin immunoprecipitation–quantitative PCR; DAPI, 4′,6-diamidino-2-phenylindole; α-dsDNA, anti–double-stranded DNA antibody (Ab); α-fortilin, anti-fortilin Ab; Fortilin WT, THP1 WT-fortilin , THP1 cells expressing WT fortilin; Fortilin KO, THP1 KO-fortilin , THP1 cells in which the fortilin genes have been deleted by the CRISPR–Cas9 technology; α-HA, anti-HA-epitope-tag antibody; IB, immunoblot; α-MEF2C, anti-MEF2C Ab; pGL-P Control , pGL-luciferase vector with no promoter; pGL-P CTNNA3 -Luc, pGL luciferase vector in which the CTNNA3 promoter is fused to the firefly luciferase construct; PLA, proximity ligation assay; Plasmid-D25A, pCS-fortilin D25A -3×HA plasmid that expresses the MEF2C-binding–deficient fortilin D25A mutant fused to the three HA epitope tag repeats at its C-terminal end; Plasmid-WT, pCS-fortilin WT -3×HA plasmid that expresses the WT fortilin fused to the three hemagglutinin (HA) epitope tag repeats at its C-terminal end; RAI, relative activity index; 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: Activation Assay, Plasmid Preparation, Binding Assay, Clone Assay, Luciferase, Reporter Assay, Transfection, Control, Cotransfection, Activity Assay, Standard Deviation, Western Blot, Knockdown, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Purification, Real-time Polymerase Chain Reaction, Positive Control, Expressing, ChIP-qPCR, CRISPR, Construct, Proximity Ligation Assay, Mutagenesis

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: Frontiers in Bioengineering and Biotechnology

Article Title: ECM proteins regulate microRNA-mediated direct reprogramming of fibroblasts into cardiomyocytes via YAP signaling

doi: 10.3389/fbioe.2026.1749865

Figure Lengend Snippet: Fibroblast reprogramming via DE-DOPE/miRcombo lipoplexes is influenced by cardiac ECM protein coatings. (a,b) Immunofluorescence images (a) and quantification (b) for Mef2C (green) in AHCFs transfected with DE-DOPE/miRcombo lipoplexes and cultured for 7 days on NC and LN, FN, CI and BM coated plates. Scale bar = 100 µm. Percentage of Mef2C positive cells was calculated by counting positive nuclei for Mef2C on total nuclei number; (c,d) Flow cytometry plots (c) and quantification (d) for cTnT-positive AHCFs transfected with DE-DOPE/miRcombo lipoplexes, after 15 days culture on NC and LN, FN, CI and BM coated plates; cTnT positive cells are shown in red; (e) Immunofluorescence images for cTnT (green) expression in cells transfected with DE-DOPE/miRcombo lipoplexes, after 15 days culture on NC and LN, FN, CI and BM coated plates. Scale bar = 100 µm. (f–h) Gene expression analysis for TNNT2 (f) , ACTC1 (g) and CACNA1C (h) using ddPCR on cells transfected with DE-DOPE/miRcombo lipoplexes, after 15 days culture on NC and LN, FN, CI and BM coated plates. Data are expressed as fold change relative to the NC condition. Data are expressed as mean ± SEM of three independent experiments. Statistical analysis was performed by 1-way ANOVA.

Article Snippet: At selected time point, AHCFs were fixed in 4% PFA (Alfa Aesar), permeabilized 0.01% Triton X 100 (Sigma-Aldrich) for 10 min, and blocked with BSA 1% in PBS for 1 h. Cells were incubated with primary antibodies against Ki67 (UM870033, UltraMAB), YAP (ab52771, abcam), Mef2C (5030, Cell Signaling), cTnT (701620, Invitrogen) and α-SAR (A7607 Sigma-Aldrich) overnight at 4 °C.

Techniques: Immunofluorescence, Transfection, Cell Culture, Flow Cytometry, Expressing, Gene Expression