primary antibodies mef2c  (Cell Signaling Technology Inc)

Journal: Nature Cardiovascular Research

Article Title: Oxidative phosphorylation is required for cardiomyocyte re-differentiation and long-term fish heart regeneration

doi: 10.1038/s44161-025-00718-x

Figure Lengend Snippet: a , Representative images of immunofluorescence staining showing Mef2 + PCNA + double-positive proliferating cardiomyocytes in TU and WIK at 7 dpci. Framed areas highlight the wound border zone (red) (scale bar, 300 μm). b , Quantification of Mef2 + PCNA + double-positive cells showing differences in proliferating cardiomyocytes in the border zone at 7 dpci but not at 21 dpci. c , Positive correlation of percentage of proliferating cardiomyocytes in the border zone between 7 dpci and 21 dpci. d , No correlation between 7 dpci border zone proliferation and 90 dpci wound length. e , Venn diagram displaying complete lack of overlap between genes correlating to 7 dpci border zone proliferation and 90-dpci wound length. f , g , No correlation between border zone proliferation and OXPHOS ( f ) or Glycolysis ( g ) at 7 dpci. h , Quantification of Mef2 + PCNA + cells showing no difference in proliferating cardiomyocytes in the border zone of 7 dpci KCL adult treated with inhibitor PF-04859989 or rotenone compared to DMSO control. i , Temporal wound length reduction of all strains between 1, 7, 21 and 90 dpci. Arrow highlighting the strong decrease in wound length in WIK between 7 dcpi and 21 dpci. j , Percentage of hearts completely regenerated at 90 dpci or with closed compact wall but remaining internal scar or with open compact wall and internal scar remaining. b , 7 dpci: AB, NA, TU n = 7; SAT, WIK n = 6; TL n = 5; KCL n = 3, 21 dpci: AB, SAT, TL, TU n = 5; NA n = 6; WIK n = 7; KCL n = 8 (biological replicates); h , PF-04859989, DMSO n = 6, rotenone n = 5 (biological replicates); i , AB: 1, 90 dpci n = 7; 7 dpci n = 8; 21 dpci n = 6. NA: 1, 7, 90 dpci n = 7; 21 dpci n = 11. SAT: 1, 7, 21, 90 dpci n = 7. TL: 1, 7, 90 dpci n = 7; 21 dpci n = 9. TU: 1, 7 dpci n = 7; 21 dpci n = 5; 90 dpci n = 6. WIK: 1, 7, 21, 90 dpci n = 7. KCL: 1, 7, 21, 90 dpci n = 8 (biological replicates); j , AB, NA, SAT, TL, WIK n = 7; TU n = 6; KCL n = 8. b , h , One-way ANOVA with Tukey’s test. c , d , f , g , Simple linear regression. h , Two-way ANOVA with Tukey’s test. i , Data presented as mean ± s.e.m. Mef2, myocyte enhancer factor 2; PCNA, proliferating cell nuclear antigen.

Article Snippet: Primary antibodies Mef2c (Biorbyt, orb256682), PCNA (clone PC10; Dako, M0879), GFP (Abcam, ab13970), MF20 (Developmental Studies Hybridoma Bank (DSHB), AB_2147781) and embcmhc N2.261 (DSHB, AB_531790) and secondary antibodies Alexa Fluor 488 (Invitrogen, anti-mouse A11001, anti-chick A11039 and anti-rabbit A21206) and Alexa Fluor 555 (Invitrogen, A31570 ) were prepared using TNB buffer at a ratio of 1:200.

Techniques: Immunofluorescence, Staining, Control

Journal: Aging (Albany NY)

Article Title: lncRNA GPRC5D-AS1 as a ceRNA inhibits skeletal muscle aging by regulating miR-520d-5p

doi: 10.18632/aging.205279

Figure Lengend Snippet: The effect of overexpression of GPRC5D-AS1 on muscle regulatory factors. ( A ) qRT-PCR analyzed gene expression of Myf5, MyoG, MyoD and Mef2c. HSMM was control group. Dex (15 mM) was added in HSMM to establish atrophy cell model (model group). Empty plasmid (NC group) and GPRC5D-AS1-OE plasmid (lncRNA-OE group) were transfected into atrophy cell model and incubated for 48 h. * P < 0.05, ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with model group; & P < 0.05, && P < 0.01 compared with NC group. ( B ) Protein expression of Myf5, MyoG, MyoD and Mef2c detected by Western blot. Groups were set as previously mentioned. Empty plasmid and GPRC5D-AS1-OE plasmid were transfected into atrophy cell model and incubated for 48 h. ( C ) Quantitative analysis of western blot. * P < 0.05, ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with model group; & P < 0.05, && P < 0.01 compared with NC group.

Article Snippet: Then, protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA), blocked with 5% skim milk and incubated with primary antibodies of anti-Mef2c (Cal.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Incubation, Western Blot

Journal: Aging (Albany NY)

Article Title: lncRNA GPRC5D-AS1 as a ceRNA inhibits skeletal muscle aging by regulating miR-520d-5p

doi: 10.18632/aging.205279

Figure Lengend Snippet: LncRNA GPRC5D-AS1 interacted with miR-520d-5p to promote myoblast proliferation. ( A , B ) qRT-PCR analyzed gene expression of GPRC5D-AS1, miR-520d-5p, MyoD1, MyoG, Mef2c, Myf5 and Wnt5a. 15 mM Dex was added in human skeletal muscle myoblasts to establish atrophy cell model (control group). Empty plasmid (NC group), GPRC5D-AS1-OE (lncRNA-OE group), GPRC5D-AS1-OE + miR-520d-5p mimic (lncRNA-OE + mimic group), miRNA inhibitor control (inhibitor NC group), miR-520d-5p inhibitor (miRNA inhibitor group) or MYOD1-OE plasmid (mRNA-OE group) was transfected into atrophy cell model and incubated for 48 h. * P < 0.05, ** P < 0.01 compared with control group; # P < 0.05, ## P < 0.01 compared with NC group; & P < 0.05, && P < 0.01 compared with lncRNA-OE group; % P < 0.05, %% P < 0.01 compared with inhibitor NC group.

Article Snippet: Then, protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA), blocked with 5% skim milk and incubated with primary antibodies of anti-Mef2c (Cal.

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Incubation

Journal: Aging (Albany NY)

Article Title: lncRNA GPRC5D-AS1 as a ceRNA inhibits skeletal muscle aging by regulating miR-520d-5p

doi: 10.18632/aging.205279

Figure Lengend Snippet: The primer sequences for mRNAs, microRNAs and long non-coding RNAs (lncRNAs).

Article Snippet: Then, protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA), blocked with 5% skim milk and incubated with primary antibodies of anti-Mef2c (Cal.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: MEF2C shapes the microtranscriptome during differentiation of skeletal muscles

doi: 10.1038/s41598-021-82706-2

Figure Lengend Snippet: The expression of muscle-specific miRNAs is correlated with MEF2C and MYOG level during skeletal muscle differentiation. ( a ) RT-PCR-based quantification of the level of miRNA, pri-miRNAs and mRNA of myogenic factors performed for primary human myoblasts in growth (day 0) or differentiation conditions (days 1, 3, 5 and 7). Image was created by using GeneTools 4.3.9.0 and CorelDRAW 2017. Fully uncut gel images are shown in Supplementary information, uncut images section; level of miR-1 and miR-133 was analyzed using TaqMan real-time PCR normalized to U6RNA. The three human primary myoblasts were tested. ( b ) Graph showing the pattern of temporal expression changes in selected pri-miRNA and their mature forms. Specific markers of different stages of muscle cells differentiation are shown above the graph. ( c ) Experimental timeline for the HSkM cell cultures. Proliferating HSkM cells were transfected with siCTRL, siMEF2C_1 or siMEF2C_2 (day 0) and then transferred to differentiation medium. The next day, the cells were transfected once again and cultured in differentiation medium for the next 3 days. ( d ) The steady state level of MEF2C protein (normalized to GAPDH) was strongly reduced during the course of the experiment. siMEF2C_1 induced more efficient reduction of MEF2C. The experiment was performed in HSkM cells in triplicates. Signals were quantified with GeneTools from Syngene ( https://www.syngene.com/software/genetools-automatic-image-analysis/ ) . Fully uncut gel images are shown in Supplementary information, uncut images section. ( e ) Three MEF2C-dependent target genes encoding muscle-specific proteins subsequently showed reduced expression on mRNA level. ( f ) The main markers of myogenesis (MYOD, MYOG and SRF) were not down-regulated in differentiated HSkM cells treated with siMEF2C_1; moderate decrease of MYOD and MYOG was observed in siMEF2C_2 KD experiments. The results in ( e ) and ( f ) are average results of RT-qPCR analyses performed for three independent samples from siMEF2C-treated (blue bars) and siCTRL-treated cells (gray bars) normalized to GAPDH. Standard deviation (SD) and statistical significance based on an unpaired Student’s t-test are shown (* P < 0.05, ** P < 0.01, *** P < 0.0001). ( g ) Fusion index of HSkM was not changed in differentiated myoblasts treated with siMEF2C_1 or siMEF2C_2. The fusion index was calculated as the number of nuclei within myotubes expressed as a percentage of the total number of nuclei in the image frame. Calculations were performed for three independent samples from siMEF2C_1 or siMEF2C_2-treated (blue bars) and siCTRL-treated cells (gray bar).

Article Snippet: MEF2C primary antibody was purchased from Cell Signaling Technology (D80C1) XP Rabbit mAb #5030.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transfection, Cell Culture, Software, Quantitative RT-PCR, Standard Deviation

Journal: Scientific Reports

Article Title: MEF2C shapes the microtranscriptome during differentiation of skeletal muscles

doi: 10.1038/s41598-021-82706-2

Figure Lengend Snippet: Changes in the expression profile of miRNAs in human skeletal myocytes with MEF2C depletion. ( a ) MA-plot showing the prediction of differentially expressed miRNAs between cells treated with control and MEF2C-specific siRNAs. The red solid dots represent the statistically significant results for miRNAs downregulated in cells treated with siMEF2C_1; the green solid dots represent significantly upregulated miRNAs by siMEF2C_1. Empty dots represent miRNAs deregulated by both siMEF2Cs (_1 and_2). The experiment was performed in HSkM cells in triplicates. ( b ) Validation of the results from ( a ) in HSkM with MEF2C KD. The expression of 14 mature miRNAs was quantified using polyA-RT-qPCR analysis. The expression level of miRNAs were normalized to the level of the small nuclear U6RNA. Green/dark green and red/orange bars represent miRNAs predicted to be upregulated or downregulated according to RNA-seq after treatment with siMEF2C_1 or siMEF2C_2, respectively. The results are average values from three independent experiments + / − SD (* P < 0.05). ( c ) Relative expression changes of pri-miRNAs quantified by RT-qPCR assays divided into two groups (miRNAs predicted to be upregulated or downregulated according to RNA-seq results); all data for pri-miRNA were averages from three independent experiments + / − SD normalized to mRNA of GAPDH (* P < 0.05). MiRNAs from the same genetic cluster are marked with a blue line. ( d ) Expression changes of miRNAs coming from the same pre-miRNA hairpin or from the same genetic cluster (marked with blue lines) based on RNA-seq data. MiRNA candidates which could be regulated transcriptionally or posttranscriptionally are indicated.

Article Snippet: MEF2C primary antibody was purchased from Cell Signaling Technology (D80C1) XP Rabbit mAb #5030.

Techniques: Expressing, Quantitative RT-PCR, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: MEF2C shapes the microtranscriptome during differentiation of skeletal muscles

doi: 10.1038/s41598-021-82706-2

Figure Lengend Snippet: MEF2C regulates 3′-uridylation of miRNAs in human muscle cells. ( a ) The pie charts show the frequency of four types of modifications of the miRNA sequence in differentiated HSkM cells. The experiment was performed in HSkM cells in triplicates. ( b ) Comparison of the frequency of 5′- and 3′-modifications of miRNAs in control and MEF2C KD cells based on the results of small RNA-seq experiments ( * P < 0.05; ** P < 0.01). ( c ) Comparison of the number and length of 3′-nontemplated U- and A-tailed reads of miRNAs in control and siMEF2C_1 or siMEF2C_2 treated myocytes. The X-axis represents the length of U- or A-tails, and the Y-axis represents the percentile of modified reads in small RNA-seq ( * P < 0.05). ( d ) Modified polyA-RT-qPCR assays were used to quantify canonical forms of miR-221 and miR-92a (lower panels) and uridylated isoforms of miR-221-U and miR-92a-U (upper panels), in muscle cells with MEF2C deficiency (* P < 0.05, ** P < 0.01, *** P < 0.0001). The experiment was performed in triplicates. ( e ) In vitro uridylation assay of internally radiolabeled pre-miR-27b incubated with extracts from siCTRL- or siMEF2C_1-treated HSkM cells. The mixtures were either supplemented or not supplemented with 0.25 mM UTP (+ or − rUTP). Reaction products were separated by denaturing PAGE, analyzed by autoradiography, and the amount of pre-miRNA short fragments (13–17 nt) was calculated and is shown on a graph (below) as a fraction of unprocessed pre-miRNA (*** P < 0.0001). Fully uncut gel images are shown in Supplementary information, uncut images section. ( f ) Accumulation of short fragments of miRNAs in differentiated HSkM cells upon siMEF2C_1 KD calculated based on RNA-seq data (n = 3 for siCTRL and n = 3 for siMEF2C_1). Alterations in the level of miRNA fragments are shown as fold changes; fold change is defined as the ratio between the counts of short fragments of miRNA in siMEF2C_1 KD and the counts of short fragments of miRNA in siCTRL cells divided by the counts of short fragments of miRNA in siCTRL cells (no change is equal to 1).

Article Snippet: MEF2C primary antibody was purchased from Cell Signaling Technology (D80C1) XP Rabbit mAb #5030.

Techniques: Sequencing, RNA Sequencing Assay, Modification, Quantitative RT-PCR, In Vitro, Incubation, Autoradiography

Journal: Scientific Reports

Article Title: MEF2C shapes the microtranscriptome during differentiation of skeletal muscles

doi: 10.1038/s41598-021-82706-2

Figure Lengend Snippet: MEF2C contributes to the 3′-uridylation of miRNAs via TUT4 activity. ( a ) Results of RT-qPCR for mRNAs encoding for TUTs and other proteins related to the turnover of miRNA (PNPT1, polyribonucleotide nucleotidyltransferase 1; RRP41, ribosomal RNA-processing protein 41; PARN poly(A)-specific ribonuclease). Each bar represents average from three independent biological replicats + / − SD normalized to GAPDH (** P < 0.01). ( b ) Reduction in the level of TUT4 protein in MEF2C KD conditions confirmed by western blotting. Quantification of TUT4 is depicted in the graph below and based on 3 independent experiments normalized to GAPDH (* P < 0.05). Signals were quantified with GeneTools from Syngene ( https://www.syngene.com/software/genetools-automatic-image-analysis/ ) . Fully uncut gel images are shown in Supplementary information, uncut images section. ( c ) RT-qPCR analysis of pre-mRNA of TUT4. The amplified region was located in intron 2 of TUT4 . The results are averages from 3 independent experiments normalized to GAPDH mRNA (= / − SD). The P value was assessed by an unpaired t -test (** P < 0.01; *** P < 0.001). ( d ) Normalized luciferase activity calculated for HeLa cells cotransfected with genetic constructs expressing two isoforms of MEF2C (MEF2Ca1 and MEF2Ca2) or GFP and the LUC gene with either a control minimal promotor (miPLuc) or minimal promotor of LUC with upstream fragment of TUT4 gene (miPfTUT4) or minimal promotor of LUC with upstream fragment of TUT4 gene with mutations of putative MEF2C binding sites (miPmut1TUT4 or miPmut2TUT4 or miPmut1TUT4; see Table for more details). Sequence of minimal promotor is depicted in Supplementary Table . The experiment was performed in 5 replicates, and the P value was assessed by an unpaired Student’s t-test (* P < 0.05; ** P < 0.01).

Article Snippet: MEF2C primary antibody was purchased from Cell Signaling Technology (D80C1) XP Rabbit mAb #5030.

Techniques: Activity Assay, Quantitative RT-PCR, Western Blot, Software, Amplification, Luciferase, Construct, Expressing, Binding Assay, Sequencing

Journal: Scientific Reports

Article Title: MEF2C shapes the microtranscriptome during differentiation of skeletal muscles

doi: 10.1038/s41598-021-82706-2

Figure Lengend Snippet: Contribution of MEF2C to miRNA regulation in differentiated muscle cells. ( a ) MA-plot showing the prediction of differentially expressed miRNAs between proliferating HSkM cells and myotubes after 4 days of differentiation (n = 3 HSkM_0h and n = 3 HSkM_4d). The red dots represent the statistically significant results for miRNAs downregulated; the green dots represent significantly upregulated miRNAs ( P adj < 0.05; a cutoff of a baseMean > 10). ( b ) Comparison of miRNA expression changes in two sets of experiments: MEF2C KD cells versus control treated cells (separately for siMEF2C_1 and siMEF2C_2) and 4 days differentiated muscle cells versus myoblasts (HSkM differentiation). Linear regression scatter plots of miRNAs log 2FC expression values were calculated by comparison of miRNAs significantly changed upon MEF2C KD (P adj < 0.05) and miRNA significantly changed upon HSkMs differentiation (P adj < 0.05). The statistical analysis for linear regression was done by using the GraphPad Prism 8.3.0 tool. ( c ) Schematic overview of MEF2C activity in muscle cells. MEF2C, as a transcription factor, can both activate (e.g., pri-miR-133a) and repress (e.g., pri-miR-376a) miRNA expression. Interaction of MEF2C within promoter of TUT4 leads to increase of TUT4 level and normal 3′-uridylation of pre-miRNAs and miRNAs. Proper dynamics of uridylation guarantee the correct turnover of short pre-miRNA fragments. Red triangles represent downregulation, and green triangles upregulation.

Article Snippet: MEF2C primary antibody was purchased from Cell Signaling Technology (D80C1) XP Rabbit mAb #5030.

Techniques: Expressing, Activity Assay