anti mef2d mouse  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: a Mef2D is extensively disordered and predicted to form liquid-liquid phase separated condensates. The Mef2D structure predicted by Alphafold indicates a small structured domain involving the N-terminal ~100 residues and most of the transactivation domain (TAD) contains is disordered. The regions promoting formation of liquid-like droplets by the FuzDrop method are marked by blue. The β-domain (magenta) appears as an ordered motif within the disordered transactivation region. FuzDrop predictions shown on the right panels indicate high droplet-promoting probability (p DP ) in particular for regions 155-268 residues and 341-520 residues, which are predicted to spontaneously form liquid-liquid phase separated condensates. The β-domain (magenta) and its flanking regions (cyan) are predicted to serve as ordered interaction motifs within the condensate (see also Supplementary Fig. ). In addition, the β-domain region is capable of sampling a multiplicity of binding modes (MBM), indicating its sensitivity to the cellular context. b Sequences of the designed Mef2D variants. The β-domain and its flanking regions are shown for the wild-type ( wt ) Mef2D (UniProt code: Q14814; https://legacy.uniprot.org/uniprot/Q14814 ; 265-301 residues), var1 and var2 with similar β-domain dynamics (gray), var3 and var4 with mobile β-domains (green), var5 - var8 with rigid β-domains (red) as compared to wild-type Mef2D. The sequence of the β-domain is magenta, mutated residues (orange) are highlighted. c Predicted β-domain disorder of Mef2D variants. Structural disorder in the unbound state of Mef2D were computed for the full protein sequence using the Espritz method as embedded in the FuzPred program and the p D values were averaged for residues 286-292. The var3 and var4 variants (green) are above the threshold between disorder and order (p D ≥ 0.3085 ). var1 (gray) has similar, var2 (gray) has slightly more mobile β-domain than the wild-type Mef2D (black). var5 - var8 variants (red) are predicted to have more rigid β-domain than the wild-type. d Droplet landscape of the Mef2D variants. The droplet landscape shows the droplet probability (p DP ) as a function of the multiplicity of binding modes (MBM) , . The assemblies below the diagonal are likely more solid-like, those above the diagonal are more liquid like . High MBM values indicate an increased likelihood to change between liquid-like and solid-like forms, for example in case of var8 . More mobile β-domain variants ( var3 , var4 , green diamonds) exhibit increased probability to form droplets (higher p DP ), whereas more rigid β-domain variants ( var5 - var8 , red triangles) more likely form solid-like states depending on the cellular conditions (high MBM).

Article Snippet: After blocking non-specific binding sites, membranes were exposed to primary antibodies overnight at 4 °C as follows: mouse monoclonal anti-MEF2D (1:500; BD Biosciences, San Jose, CA, USA), monoclonal mouse myoD (Novus Biologicals, Abington, UK) antibody in 1:200 dilution, mouse monoclonal anti-desmin in 1:1000 dilution (BD Biosciences) and goat polyclonal anti-actin (1:250; Santa Cruz Biotechnology, Dallas, USA).

Techniques: Sampling, Binding Assay, Sequencing

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: Luciferase activity normalised to galactosidase signal (Methods) is shown as a percentage of the wild-type (wt) control. Luciferase activity was measured in four biologically independent experiments, using three technical replicates in each with the same samples ( n = 12 samples in 4 independent experiments). The points represent individual measured data, the rectangles in the box plots present the median and the 25 and 75 percentile values, while the error bars point to 1 and 99%. The luciferase signal is shown as mean ± SE, significance (* p < 0.05; ** p < 0.01; # p < 0.005; ## p < 0.001) was computed using two-sided student t-test. The different variants are grouped by their β-domain dynamics properties (Fig. , Methods): var1 (gray diamond) and var2 (gray triangle) with similar β-domain dynamics; var3 (green diamond) and var4 (green triangle) with mobile β-domain; var5 (red diamond), var6 (red triangle), var7 (red circle) and var8 (red square) with rigid β-domain as compared to the wild-type. The β- variant is shown by blue diamond. a Transcriptional activity in non-differentiated C2C12 myoblasts. Variants with rigid β-domains show significantly higher transcriptional activity then the wild-type ( var5 178 ± 9.6, var6 135.6 ± 9.3, var7 141.6±8.1 and var8 140.8±11.5 %), while variants with mobile β-domains show slightly increased transcription activity ( var3 127.7 ± 4.9 and var4 123.9 ± 5.5 %) using n = 12 samples in 4 independent experiments. Significances ( var3 p = 0.0001, var4 p = 0.0012, var5 p = 5.2*10 −6 , var6 p = 0.0027, var7 p = 0.0003, var8 p = 0.0044; β-minus p = 1.52*10 −6 ) were computed using two-sided student t-test. b Transcriptional activity in differentiated C2C12 myotubes . var8 with rigid β-domain (red square) exhibits significantly higher transcriptional activity than the wild-type, while var3 (green diamond) and var4 (green triangle) with mobile β-domains, and var2 (gray triangle) with similar β-domain dynamics as the wild-type exhibit reduced transcriptional activity. ( n = 12 samples in 4 independent experiments). Significances ( var2 p = 2.9*10 −9 ; var3 p = 0.008; var4 p = 1.02*10 −7 ; var8 p = 0.0004) were computed using two-sided student t-test. c Lack of Mef2D blocks myotube formation in Mef2D knockout C2C12 cell line. Western blot images showing the lack of Mef2D, which are present endogenously in C2C12 cells. Three chosen stable KO cultures were followed through several passages to prove the stable lack of Mef2D (~70 kDa), while actin was used as inner control (~40 kDa). After 6 days of differentiation myotubes form in C2C12 cell line with endogeneous Mef2D (control, left panel), while cannot be observed in MEF2D KO cultures (right panel). Images were taken by transmitted microscopy, scale bars represent 400 µm. d Transcriptional activity in C2C12 KO cells. Rigid β-domain variants (red) have higher transcriptional activity than variants with similar dynamics to the wild-type (gray). Significances ( var4 p = 0.0175, var5 p = 0.0096, var6 p = 0.0365; var7 p = 0.0022; var8 p = 0.0071) were computed using two-sided student t-test using n = 9 samples in 3 independent experiments).

Article Snippet: After blocking non-specific binding sites, membranes were exposed to primary antibodies overnight at 4 °C as follows: mouse monoclonal anti-MEF2D (1:500; BD Biosciences, San Jose, CA, USA), monoclonal mouse myoD (Novus Biologicals, Abington, UK) antibody in 1:200 dilution, mouse monoclonal anti-desmin in 1:1000 dilution (BD Biosciences) and goat polyclonal anti-actin (1:250; Santa Cruz Biotechnology, Dallas, USA).

Techniques: Luciferase, Activity Assay, Variant Assay, Knock-Out, Western Blot, Microscopy

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: a Early stage of myotube development (day 2 - day 4). The number of multinucleated, long myotubes in the presence of overexpressed Mef2D variants. Representative fluorescent and transmitted images represent randomly selected visual fields and were used to determine the fusion index of the appropriate cultures. Each experiment was independently repeated two times with similar results, at least 15 randomly selected visual fields were analysed. Scale bar is 50 µm. b , c Protein expression of myogenic regulatory factors MyoD ( b ) and Desmin ( c ). Normalized protein expression during the differentiation of C2C12 cells. Protein expression was plotted as a percentage of their wild-type control in each day of differentiation. Data was derived from quadruplicate measurements ( n = 4 independent experiments), significances (* p = 0.0032; ** p = 0.0172 for panel b , and * p = 0.0149; ** p = 0.0002 for panel c ) were computed using two-sided student t-test as compared to the wild-type control on the given day of differentiation. b Protein expression of the early differentiation regulator MyoD. Variants with rigid β-domains ( var5 - var8 , red) exhibit higher level of MyoD expression on day 1 and day 2. Significant deviations ( var5 p = 0.0032; var8 p = 0.0172) were observed in case of var5 and var8 using two-sided student t-test as compared to the wild-type control on the given day of differentiation. c Protein expression of the late differentiation marker desmin. More rigid β-domain variants ( var5 p = 0.0149; var8 p = 0.0002, red) significantly increase desmin expression on days 1 and 2.

Article Snippet: After blocking non-specific binding sites, membranes were exposed to primary antibodies overnight at 4 °C as follows: mouse monoclonal anti-MEF2D (1:500; BD Biosciences, San Jose, CA, USA), monoclonal mouse myoD (Novus Biologicals, Abington, UK) antibody in 1:200 dilution, mouse monoclonal anti-desmin in 1:1000 dilution (BD Biosciences) and goat polyclonal anti-actin (1:250; Santa Cruz Biotechnology, Dallas, USA).

Techniques: Expressing, Derivative Assay, Marker

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: a Mef2D foci in the nucleus and cytoplasm. Subcellular distribution of Mef2D wt , var3 , var4 and var8 in C2C12 cells grown in cycling or differentiating medium exhibit foci formation in both the nucleus and cytoplasm. Higher-order assembly is most pronounced in case of var8 with rigid β-domain, but is also observed in case of var3 and var4 with mobile β-domain. 24 hrs post-transfection cells were fixed and stained with an antibody specific for Mef2D. Nucleic acid was stained using DAPI. The scale bar is 10 μm on the representative images. The experiment was performed four times (cycling medium) and three times (differentiating medium). Quantification is shown in panel b . b Quantification of MEF2D cells with cytoplasmic higher-order structures (foci). The percentage of Mef2D overexpressing cells with cytoplasmic aggregates is significantly higher in case of var8 with rigid β-domain. Cycling C2C12 cells: n = 4, ± s.e.m.; differentiating C2C12 cells: n = 3 independent experiments, ± s.e.m. Total number of cells counted > 150. Significances were computed by one-way ANOVA followed by Bonferroni-Holm Posthoc in reference to wt : var3 p = 0.13 (cycling) and p = 0.87 (differentiated), var4 p = 0.09 (cycling) and p = 0.60 (differentiated), var8 p = 0.04 (cycling) and p = 0.04 (differentiated). c , d Analysis of mobility of Mef2D higher-order assemblies in nuclear foci ( c ) and cytoplasmic foci ( d ). Mobility was assessed by fluorescence recovery after photobleaching (FRAP) performed after 24 hours post-transfection of GFP-tagged MEF2D wt, var3 , var4 and var8 in C2C12 cells. The mean of the FRAP curve +/- standard error of the mean (s.e.m.) is shown. c Number of nuclear foci analyzed: wt (3); var3 (3); var4 (3); var8 (3). d number of cytoplasmic aggregates analyzed: wt (11); var3 (10); var4 (10); var8 (9). All Mef2D proteins show high mobility inside the nuclear foci ( c ) and nucleoplasm (Supplementary Fig. ). This is in sharp contrast with the low mobility inside cytoplasmic foci ( d ).

Article Snippet: After blocking non-specific binding sites, membranes were exposed to primary antibodies overnight at 4 °C as follows: mouse monoclonal anti-MEF2D (1:500; BD Biosciences, San Jose, CA, USA), monoclonal mouse myoD (Novus Biologicals, Abington, UK) antibody in 1:200 dilution, mouse monoclonal anti-desmin in 1:1000 dilution (BD Biosciences) and goat polyclonal anti-actin (1:250; Santa Cruz Biotechnology, Dallas, USA).

Techniques: Transfection, Staining, Fluorescence

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: Conformational analysis was performed using the 70-100 ns trajectory of each replica (9000 snapshots) (Methods). a Contacts maps of the clusters. Mef2D wt , var3 , var4 and var8 peptides exhibit distinct intra-molecular interaction patterns (Methods). The β-domain (blue) contributes to structure organisation of var8 and to lesser extent to var4 , while does not form persisting contacts in var3 (see also Supplementary Fig. ). Color scales indicate the number of snapshots in the clusters, with the given contact sampled. b Representative structures of the of Mef2D variant peptides. More compact structures are formed through interactions of the β-domain (blue), such as in case of var8 and var4 , while extended structures, such as in case of var3 sample variable interactions outside the β-domain. β-domain residues are displayed in blue, residues mutated in the different variants are orange labelled.

Article Snippet: After blocking non-specific binding sites, membranes were exposed to primary antibodies overnight at 4 °C as follows: mouse monoclonal anti-MEF2D (1:500; BD Biosciences, San Jose, CA, USA), monoclonal mouse myoD (Novus Biologicals, Abington, UK) antibody in 1:200 dilution, mouse monoclonal anti-desmin in 1:1000 dilution (BD Biosciences) and goat polyclonal anti-actin (1:250; Santa Cruz Biotechnology, Dallas, USA).

Techniques: Variant Assay

Journal: Cell Regeneration

Article Title: Myogenesis controlled by a long non-coding RNA 1700113A16RIK and post-transcriptional regulation

doi: 10.1186/s13619-022-00114-x

Figure Lengend Snippet: 1700113A16Rik promoted MuSCs differentiation by targeting MEF2D. A The locations of 1700113A16Rik and MEF2D in the genome. The 3’UTR of Mef2d complementary paired with 1700113A16RIK. B The relative RNA level of 1700113A16Rik in the differentiated MuSCs. MuSCs were transfected by two pieces of siRNA against 1700113A16Rik (si1700113A16Rik-1 and si1700113A16Rik-2) or scramble RNA (siNC) and then differentiated for 3 days. RT-qPCR assays were performed with RNA extracted from the differentiated MuSCs in the control group and the 1700113A16Rik knockdown group. Error bars indicated standard deviation and were based on 3 independent experiments. * indicated p <0.05, ** indicated p <0.01. C The relative RNA level of Mef2d in the differentiated MuSCs described in B . RT-qPCR assays were performed with RNA extracted from the differentiated MuSCs in the control group and the 1700113A16Rik knockdown group. Error bars indicated standard deviation and were based on 3 independent experiments. NS indicated no significant changes. D The protein level of MEF2D in the differentiated MuSCs described in B . Western blot was performed from the differentiated MuSCs in the control group and the 1700113A16Rik knockdown group. E The relative RNA level of 1700113A16Rik in the differentiated MuSCs. MuSCs were infected by the control adenovirus and adenovirus encoding 1700113A16Rik and then differentiated for 3 days. RT-qPCR assays were performed with RNA extracted from the differentiated MuSCs in the control group and the 1700113A16Rikoverexpressing group. Error bars indicated standard deviation and were based on 3 independent experiments. *** indicated p <0.001. F The relative RNA level of Mef2d in the differentiated MuSCs described in E . RT-qPCR assays were performed with RNA extracted from the differentiated MuSCs in the control group and the 1700113A16Rik-overexpressing group. Error bars indicated standard deviation and were based on 3 independent experiments. NS indicated no significant changes. G The protein level of MEF2D in the differentiated MuSCs described in E . Western blot was performed from the differentiated MuSCs in the control group and the 1700113A16Rik-overexpressing group. The numbers below each panel indicated the relative signal intensity. H The luciferase activity of reporter gene carrying 1700113A16Rik target sequence from Mef2d at the 3’UTR region. Firefly luciferase activity regulated 1700113A16Rik target sequence at the 3’UTR. Renilla luciferase activity driven by CMV promoter served as an internal control. Error bars indicated standard deviation and were based on 5 independent experiments. * indicated p <0.05. NS indicated no significant changes

Article Snippet: The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with anti-MyHC (Upstate, 05-716), anti-GAPDH (Cell signaling technology, 2118) antibodies, mouse anti-Mef2d (BD, 610774) at 4 °C overnight.

Techniques: Transfection, Quantitative RT-PCR, Standard Deviation, Western Blot, Infection, Luciferase, Activity Assay, Sequencing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Myocyte-specific enhancer factor 2D promotes tumorigenesis and progression in tongue squamous cell carcinoma

doi:

Figure Lengend Snippet: Association between MEF2D expression and clinicopathologic features in TSCC patients

Article Snippet: Subsequent to blocking with 5% skim milk for 1 hour at room temperature, membranes were incubated with primary mouse monoclonal antibodies against human MEF2D (1:2000 dilution; Abcam, USA) and GAPDH (as an internal control, 1:500 dilution; bioPM, China) on a shaker at 4°C overnight.

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Myocyte-specific enhancer factor 2D promotes tumorigenesis and progression in tongue squamous cell carcinoma

doi:

Figure Lengend Snippet: MEF2D is overexpressed in TSCC patient tissues. A. Expression of MEF2D mRNA in ten, randomly selected, paired TSCC samples assessed by RT-qPCR. B. Expressions of MEF2D protein in ten, randomly selected, paired TSCC samples analyzed by WB.

Article Snippet: Subsequent to blocking with 5% skim milk for 1 hour at room temperature, membranes were incubated with primary mouse monoclonal antibodies against human MEF2D (1:2000 dilution; Abcam, USA) and GAPDH (as an internal control, 1:500 dilution; bioPM, China) on a shaker at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Myocyte-specific enhancer factor 2D promotes tumorigenesis and progression in tongue squamous cell carcinoma

doi:

Figure Lengend Snippet: Expression of MEF2D in TSCC patients. A. Statistical analysis of MEF2D expression and the degree of tumor differentiation. B. Typical IHC staining for MEF2D expression in consecutive paraffin sections (bar = 50 μm).

Article Snippet: Subsequent to blocking with 5% skim milk for 1 hour at room temperature, membranes were incubated with primary mouse monoclonal antibodies against human MEF2D (1:2000 dilution; Abcam, USA) and GAPDH (as an internal control, 1:500 dilution; bioPM, China) on a shaker at 4°C overnight.

Techniques: Expressing, Immunohistochemistry

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Myocyte-specific enhancer factor 2D promotes tumorigenesis and progression in tongue squamous cell carcinoma

doi:

Figure Lengend Snippet: Proliferation of MEF2D-silenced TTSCCA cells was determined by the CCK-8 assay.

Article Snippet: Subsequent to blocking with 5% skim milk for 1 hour at room temperature, membranes were incubated with primary mouse monoclonal antibodies against human MEF2D (1:2000 dilution; Abcam, USA) and GAPDH (as an internal control, 1:500 dilution; bioPM, China) on a shaker at 4°C overnight.

Techniques: CCK-8 Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Myocyte-specific enhancer factor 2D promotes tumorigenesis and progression in tongue squamous cell carcinoma

doi:

Figure Lengend Snippet: Suppression of MEF2D resulted in a decrease in the invasive ability of TTSCCA cells as measured by Transwell assay. *P<0.05.

Article Snippet: Subsequent to blocking with 5% skim milk for 1 hour at room temperature, membranes were incubated with primary mouse monoclonal antibodies against human MEF2D (1:2000 dilution; Abcam, USA) and GAPDH (as an internal control, 1:500 dilution; bioPM, China) on a shaker at 4°C overnight.

Techniques: Transwell Assay