Journal:

Article Title: A retrograde neuronal survival response: Target-derived neurotrophins regulate MEF2D and bcl-w

doi: 10.1523/JNEUROSCI.0233-09.2009

Figure Lengend Snippet: (A) MEF2D protein is detected in vivo in dorsal root ganglia at E18 (Lanes 1 and 2 represent two separate animals). As a positive control, lysates of neurons over-expressing MEF2VP16 were blotted for MEF2D. (B) MEF2D immunostaining of dorsal root ganglia at P0 in vivo. Scale bar = 10 μm (C) Expression of mef2d mRNA in DRG neurons in response to neurotrophin (NT) stimulation. DRG neurons were treated with neurotrophins (100 ng/ml NGF+BDNF) for 2 hours either on distal axons (DA) or on cell bodies (CB). Mef2D mRNA expression is specifically induced by distal axon stimulation and not by cell body stimulation. Expression is compared to DRG neurons treated with vehicle (100 ng/ml BSA). Results represent the mean ± SEM of 8 experiments, *P<0.05. (D) Mef2d mRNA expression is upregulated upon stimulation of distal axons with NGF alone, BDNF alone or NGF+BDNF (NT). Induction of cfos mRNA is shown as a positive control. (E) MEF2D protein levels were analyzed by Western blotting before and after NT stimulation for 2 hours. Normalized relative band density of MEF2D/actin reveal an increase of 21% ± 7% in response to distal axon stimulation.

Article Snippet: Quantitative real-time RT PCR was performed using Taqman Gene expression assays (Applied Biosystems) to assess the expression of c-fos (Rn02105452_s1), bcl-w (Rn00821025_s1), alsin (Mm00511865_m1), igf-1 (Rn00710306_m1), decorin (Rn01503161_m1), enpep (Rn00573861_m1) and mef2d (Rn00578329_m1).

Techniques: In Vivo, Positive Control, Expressing, Immunostaining, Western Blot

Journal:

Article Title: A retrograde neuronal survival response: Target-derived neurotrophins regulate MEF2D and bcl-w

doi: 10.1523/JNEUROSCI.0233-09.2009

Figure Lengend Snippet: (A) DRG neurons in culture were infected with lentivirus expressing mef2d-GFP RNAi and cell lysates analyzed by Western blotting with indicated antibodies. Mef2d RNAi (+) results in decreased expression of MEF2D protein levels while a control lentivirus (−) does not. Cerebellar lysate (Cb lysate) was run as a positive control. (B) Apoptosis of DRG neurons was analyzed by TUNEL staining. Mef2d RNAi reduces survival of DRG neurons in response to neurotrophins when applied to cell bodies and blocks survival when applied to distal axons. Results represent the mean ± SEM of 5 experiments, *P<0.05. (C) Representative images of DRG neuron cell bodies grown in culture and stimulated with neurotrophins or vehicle control on cell bodies (CB) or on distal axons (DA). Cells with (mef2d RNAi) or without (control RNAi) mef2d reduction are shown. Panels show TUNEL staining (red) and DAPI-stained nuclei in control (top) and in neurons expressing mef2d RNAi (bottom).

Article Snippet: Quantitative real-time RT PCR was performed using Taqman Gene expression assays (Applied Biosystems) to assess the expression of c-fos (Rn02105452_s1), bcl-w (Rn00821025_s1), alsin (Mm00511865_m1), igf-1 (Rn00710306_m1), decorin (Rn01503161_m1), enpep (Rn00573861_m1) and mef2d (Rn00578329_m1).

Techniques: Infection, Expressing, Western Blot, Control, Positive Control, TUNEL Assay, Staining

Journal:

Article Title: A retrograde neuronal survival response: Target-derived neurotrophins regulate MEF2D and bcl-w

doi: 10.1523/JNEUROSCI.0233-09.2009

Figure Lengend Snippet: (A) MEF2D is phosphorylated in response to ERK5 activation. COS cells were transfected with CA-MEK5 and WT-ERK5, or WT-ERK5 alone and lysates were blotted with an antibody to phospho-MEF2 (p-MEF2). (B) MEF2D is phosphorylated in response to cell body stimulation (20% ± 8% increase, P<0.05) for 20 minutes. Lysates were blotted with an antibody to phospho-MEF2 and with an antibody to pan-actin as a loading control. (C) MEF2D is phosphorylated in response to distal axon stimulation (17% ± 6% increase, P<0.05) for 2 hours. Lysates were blotted with an antibody to phospho-MEF2 and with an antibody to pan-actin as a loading control. (D) Neurons were infected with an adenovirus that expresses HA-tagged ERK5-dominant negative form (DN-ERK5), then treated with neurotrophins for 30 minutes. Compared to uninfected neurons, expression of DN-ERK5 prevents ERK5 phosphorylation without inhibiting ERK1/2 activation. (E) ERK5 supports neuronal survival induced by target-derived neurotrophins. Cell apoptosis was measured by TUNEL staining in uninfected neurons, and in neurons infected with WT-ERK5 or DN-ERK5 for two days. DN-ERK5 blocks survival of neurons that depend on target-derived neurotrophins, whereas DN-ERK5 has a lesser effect on survival supported by cell body stimulation. All data shown are means ± SEM, * P ≤ 0.05.

Article Snippet: Quantitative real-time RT PCR was performed using Taqman Gene expression assays (Applied Biosystems) to assess the expression of c-fos (Rn02105452_s1), bcl-w (Rn00821025_s1), alsin (Mm00511865_m1), igf-1 (Rn00710306_m1), decorin (Rn01503161_m1), enpep (Rn00573861_m1) and mef2d (Rn00578329_m1).

Techniques: Activation Assay, Transfection, Control, Infection, Dominant Negative Mutation, Expressing, Phospho-proteomics, Derivative Assay, TUNEL Assay, Staining

Journal:

Article Title: A retrograde neuronal survival response: Target-derived neurotrophins regulate MEF2D and bcl-w

doi: 10.1523/JNEUROSCI.0233-09.2009

Figure Lengend Snippet: (A) Expression of bcl-w mRNA in response to neurotrophin stimulation. DRG neurons were treated with neurotrophin (NT=100 ng/ml NGF+BDNF) for 2 hours either on distal axons (DA) or on cell bodies (CB). Bcl-w mRNA expression is specifically induced by distal axon stimulation and not by cell body stimulation. Bcl-w mRNA is also upregulated upon distal axon stimulation with NGF or BDNF alone. All data show mean ± SEM, *P<0.05. (B) Compartmented cultures were stimulated at cell bodies (CB) or distal axons (DA) for 8 hours with neurotrophins. Cell body lysates were analyzed by Western blotting for Bcl-w (recombinant-Bclw was the positive control). DA stimulation leads to an increase in Bcl-w protein levels (32% ± 5%). (C) The Trk kinase inhibitor K252a or vehicle control was applied to cell bodies and distal axons at 200 nM, a concentration that inhibits phosphorylation of Trk receptors in these neurons. Neurons in compartmented cultures were globally treated or not with K252a and distal axons (DA) were stimulated with neurotrophins. K252a prevents induction of c-fos, bcl-w and mef2d by target-derived neurotrophins. (D) The Erk kinase inhibitor UO126 or vehicle control was applied to cell bodies and distal axons at 50 nM, a concentration that inhibits phosphorylation of ERK kinases. Neurons in compartmented cultures were treated or not with UO126. UO126 prevents bcl-w, mef2d and c-fos induction by target-derived neurotrophins. Neurons were infected with DN-MEK5 adenovirus for three days, then distal axons were stimulated with neurotrophins. DN-MEK5 expression inhibits bcl-w and mef2d mRNA induction but not c-fos induction by target-derived neurotrophins. (E) Lentivirus expressing mef2d RNAi (M2) inhibits induction of bcl-w and mef2d, but not induction of c-fos, in response to target-derived neurotrophins (C=control lentivirus). All data show means ± SEM, *P<0.05. (F) Bcl-w and mef2d mRNA expression is specifically induced by distal axon stimulation and not by global stimulation (cultures maintained for 5–7 days).

Article Snippet: Quantitative real-time RT PCR was performed using Taqman Gene expression assays (Applied Biosystems) to assess the expression of c-fos (Rn02105452_s1), bcl-w (Rn00821025_s1), alsin (Mm00511865_m1), igf-1 (Rn00710306_m1), decorin (Rn01503161_m1), enpep (Rn00573861_m1) and mef2d (Rn00578329_m1).

Techniques: Expressing, Western Blot, Recombinant, Positive Control, Control, Concentration Assay, Phospho-proteomics, Derivative Assay, Infection

Journal:

Article Title: A retrograde neuronal survival response: Target-derived neurotrophins regulate MEF2D and bcl-w

doi: 10.1523/JNEUROSCI.0233-09.2009

Figure Lengend Snippet: (A) Representative images of DAPI staining in dorsal root ganglia from cervical and lumbar regions of the spinal cord in WT (+/+) and bcl-w −/− mice. Arrows indicate apoptotic cells. Scale bar = 10 μm (B) In vivo analysis of apoptosis. Bcl-w −/− P0 mice show an increase in cell death compared to WT littermates, in both cervical and lumbar DRGs. The percentage of cells in the DRG with condensed nuclei when visualized by DAPI staining are shown. Three animals of each genotype were used, and 3–6 dorsal root ganglia from each lumbar and cervical region were counted. All data show means ± SEM, *P<0.0001. (C) Survival of bclw−/− sensory neurons in compartmented cultures stimulated with neurotrophin applied to the distal axons (DA) or the cell bodies and proximal axons (CB). DRG neurons were dissected at E15 and seeded in compartmented cultures. Bcl-w −/− neurons are more prone to apoptosis in serum free-media compared to bcl-w +/+ and +/− under all conditions tested, *P < 0.05 (D) Retrograde response genes: DRG neurons in compartmented cultures were stimulated with NGF and BDNF or vehicle applied either to distal axons (DA), or the cell bodies and proximal axons (CB), for 2 hours. RNA was prepared and expression of decorin, alsin, enpeptidase, mef2d, IGF-1, bcl-w and c-fos were assessed by Q-RT-PCR and normalized to gapdh in the same sample. Fold induction was measured by comparing normalized level of expression in neurotrophin-treated/vehicle treated cells for 4 experiments each involving 5 cultures for each condition (DA shown first in black). Statistical analysis by Z-test, **P<0.05, *P<0.10 for a difference from 1.

Article Snippet: Quantitative real-time RT PCR was performed using Taqman Gene expression assays (Applied Biosystems) to assess the expression of c-fos (Rn02105452_s1), bcl-w (Rn00821025_s1), alsin (Mm00511865_m1), igf-1 (Rn00710306_m1), decorin (Rn01503161_m1), enpep (Rn00573861_m1) and mef2d (Rn00578329_m1).

Techniques: Staining, In Vivo, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: A retrograde neuronal survival response: Target-derived neurotrophins regulate MEF2D and bcl-w

doi: 10.1523/JNEUROSCI.0233-09.2009

Figure Lengend Snippet: (A) Neurotrophin binding and Trk activation results in endocytosis of the activated receptor complex. (B) The signaling endosome, including downstream messengers, is retrogradely transported by microtubule dependent dynein motors. (C) Upon reaching the cell body, phosphorylated ERK5 translocates to the nucleus. (D) MEF2D is phosphorylated and activated in response to a retrograde ERK5 signal. (E) Activated MEF2D initiates transcription of bcl-w, mef2d and other retrograde response genes (RRGs) to promote survival mediated by target-derived neurotrophins. (F) In the case of cell body neurotrophin stimulation, both ERK5 and ERK1/2 are activated, however, expression of bcl-w, mef2d and other retrograde response genes are not induced. One possible mechanism is through suppression of ERK5 by the ERK1/2 pathway.

Article Snippet: Quantitative real-time RT PCR was performed using Taqman Gene expression assays (Applied Biosystems) to assess the expression of c-fos (Rn02105452_s1), bcl-w (Rn00821025_s1), alsin (Mm00511865_m1), igf-1 (Rn00710306_m1), decorin (Rn01503161_m1), enpep (Rn00573861_m1) and mef2d (Rn00578329_m1).

Techniques: Binding Assay, Activation Assay, Derivative Assay, Expressing

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: Chromatin IP was performed using anti-MEF2D antibody (NBP-1-80567, Novus Biologicals) with overnight incubation at 4 °C.

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: Chromatin IP was performed using anti-MEF2D antibody (NBP-1-80567, Novus Biologicals) with overnight incubation at 4 °C.

Techniques: Luciferase, Activity Assay, Control, 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: Chromatin IP was performed using anti-MEF2D antibody (NBP-1-80567, Novus Biologicals) with overnight incubation at 4 °C.

Techniques: Expressing, Control, 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: Chromatin IP was performed using anti-MEF2D antibody (NBP-1-80567, Novus Biologicals) with overnight incubation at 4 °C.

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: Chromatin IP was performed using anti-MEF2D antibody (NBP-1-80567, Novus Biologicals) with overnight incubation at 4 °C.

Techniques: Variant Assay

Journal:

Article Title: A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation

doi: 10.1101/gad.1234304

Figure Lengend Snippet: The p38 pathway and Mef2D are rate-limiting for late-stage genes. (A) Examples of simple network motifs based on Lee et al. (2002). In a single-input motif, factor A directly regulates the three targets B, C, and D. The simple cascade depicts sequential activation with only gene B directly activated by A. In the feed-forward loop, A directly regulates each gene and sequential activation is achieved by requiring both A and B to express gene C, and both A and C to express gene D. (B) MDER fibroblasts were induced to differentiate for varying times and analyzed by Northern and Western blot. (SB) Treatment of cells with SB203580; (CIP) phosphatase treatment prior to SDS-PAGE. (C) Ratio of the average gene expression in each temporal cluster of MyoD-activated genes comparing cells constitutively expressing MKK6E or treated with SB203580 with the expression in untreated cells, demonstrating effects of p38 activity on clusters of genes activated at different stages of differentiation. Ratios are in log2 space; a value of 0 indicates no treatment change, a value of 1 indicates a twofold increase due to the treatment, and a value of -1 indicates a twofold reduction. Microarray expression data was generated from MDER cells induced to differentiate for 24 h with MKK6E-expressing virus or control virus, and analyzed together with previously generated data for control and SB203580-treated MDER cells (Bergstrom et al. 2002). Clusters 1-6 are the earliest to latest activated clusters, and cluster 7 is a transiently expressed group of genes (Bergstrom et al. 2002). Error bars represent standard error, except cluster 7, where error bars are absent because only one gene from this cluster was affected. ANOVA p-value < 0.0001 for both MKK6E and SB203580. Post-hoc testing indicated that cluster 6 differs significantly from the other clusters in response to MKK6E. (D) MDER cells were infected with the indicated retroviruses, induced to differentiate for 12 h, and subjected to Northern analysis for late-stage genes Myh3 and Des and the early-stage gene Cdh15. Mef2 is the Mef2D isoform, Con is an empty control retrovirus.

Article Snippet: ChIP, Western, immunofluorescence, and antibodies Antibodies used for ChIP were as follows: Mef2A (Santa Cruz), note that this antibody also recognizes Mef2C and Mef2D (B.H.

Techniques: Activation Assay, Northern Blot, Western Blot, SDS Page, Expressing, Activity Assay, Microarray, Generated, Infection

Journal:

Article Title: A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation

doi: 10.1101/gad.1234304

Figure Lengend Snippet: Mef2D and p38 are rate limiting for the activation of late-stage genes. (A) MDER cells were infected with MKK6E and/or Mef2D-expressing retrovirus as indicated, induced to differentiate for varying lengths of time, and analyzed by Northern blot with the indicated probes. GFP is used as a control retrovirus. (B) MDER fibroblasts were transfected with the MKK6E and/or Mef2D retrovirus as indicated, induced to differentiate, and examined by immunofluorescence for the indicated late-stage (Myh3 and Des) and early-stage (Mgn) gene products. The bars indicate the average number of positive cells per field.

Article Snippet: ChIP, Western, immunofluorescence, and antibodies Antibodies used for ChIP were as follows: Mef2A (Santa Cruz), note that this antibody also recognizes Mef2C and Mef2D (B.H.

Techniques: Activation Assay, Infection, Expressing, Northern Blot, Transfection, Immunofluorescence

Journal:

Article Title: A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation

doi: 10.1101/gad.1234304

Figure Lengend Snippet: p38 regulates transcription factor recruitment. (A) MDER fibroblasts were either untreated (0 h) or infected with MKK6E retrovirus (+) or empty vector control retrovirus (-) and induced to differentiate for 12, 24, or 48 h. ChIP was performed with broad-specificity Mef2 antiserum, and multiplex PCR for the indicated promoters was performed with pancreatic amylase (Amy) as an internal control. Input chromatin was amplified over a 30-fold range to verify linearity of the assay. Graphs indicate IP promoter fold enrichment relative to input chromatin. Data for a representative ChIP are shown with error bars indicating S.E.M. for triplicate PCR. (B) Mef2 ChIP demonstrating that active MyoD is necessary for Mef2 binding, and that the broad-specificity Mef2 antisera identifies Mef2 binding both with and without exogenous Mef2D expression. (NS) Nonspecific IgG control ChIP; (E2) β-estradiol to induce MyoD activity. (C) ChIP using Mef2D monoclonal antibody shows Mef2D is not bound at early times in the absence of exogenous Mef2D. (GFP) A control retrovirus; (K6 + 2D or 6 + D) a combination of the MKK6E and Mef2D retroviruses. (D) ChIP using MyoD antisera shows p38 regulation of MyoD binding but no dependence on Mef2D. Input titration shown in C. (Empty) Control virus without effector gene.

Article Snippet: ChIP, Western, immunofluorescence, and antibodies Antibodies used for ChIP were as follows: Mef2A (Santa Cruz), note that this antibody also recognizes Mef2C and Mef2D (B.H.

Techniques: Infection, Plasmid Preparation, Multiplex Assay, Amplification, Binding Assay, Expressing, Activity Assay, Titration

Journal:

Article Title: A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation

doi: 10.1101/gad.1234304

Figure Lengend Snippet: Mef2D and p38 regulate Pol II recruitment and progression. (A) MDER cells were transduced with control (GFP), MKK6E (K6), and/or Mef2D (2D) retroviruses, induced to differentiate 12 h and ChIP for Pol II, or P-CTD Pol II performed. The combination of Mef2D and p38 is associated with Pol II recruitment and phosphorylation. (B) MDER cells were transduced with the indicated retroviruses and induced to differentiate for 16 h. At this later time point when endogenous Mef2D and other muscle-specific Mef2 isoforms are accumulating, p38 activation induces polymerase progression and accumularion of P-CTD Pol II in the 3′ end of the gene. ChIP performed for Pol II and Ser 5-phosphorylated Pol II (P-CTD Pol II). Amplification of the gene promoter and a region in the 3′-transcribed regions of the gene (exon 7 of desmin, 6.5 kb from the promoter, and exon 27 of MHC, 19 kb from the promoter) was performed. Titrations, internal control, and graphs are as previously indicated.

Article Snippet: ChIP, Western, immunofluorescence, and antibodies Antibodies used for ChIP were as follows: Mef2A (Santa Cruz), note that this antibody also recognizes Mef2C and Mef2D (B.H.

Techniques: Transduction, Activation Assay, Amplification

Journal: Nutrients

Article Title: Programming Skeletal Muscle Metabolic Flexibility in Offspring of Male Rats in Response to Maternal Consumption of Slow Digesting Carbohydrates during Pregnancy

doi: 10.3390/nu12020528

Figure Lengend Snippet: Muscle functionality in the offspring. Myosin content and grip strength were determined to address muscle functionality. MEF2D was measured as a marker of muscle differentiation at weaning ( W ) and adolescence ( A ). Values are means ± SEM ( n = 8 for each experimental group). HF/SD: offspring from mothers on high-fat diet containing slow digesting carbohydrates; HF/RD: offspring from mothers on high-fat diet containing rapid digesting carbohydrates; Ref: offspring from mothers on AIN93G diet. * Significant difference with HF/SD group, p < 0.05. # Significant difference with reference group, p < 0.05.

Article Snippet: Specific antibodies against GLUT4 (Biogenesis Ltd., Poole, UK); myosin heavy chain F59 (Developmental Studies Hybridoma Bank, Iowa City, Iowa); total and phospho (Ser473)-PKB/Akt, total and phospho (Thr202/Tyr204)-p44/42 MAPK (ERK1/2), total and phospho (Thr172)-AMPKα2, total and phospho (Ser2448)-mTOR and MEF2D (Cell Signaling, Beverly, MA, USA); GLUT1, PKM2, PDK4, ATPase5B and UCP2 (Santa Cruz Biotechnology (Dallas, TX, USA); CD36/SR-B3 (Novus Biologicals, Centennial, CO, USA) were used. β-actin (Sigma-Aldrich, Saint Louis, MO, USA) was used as a load control.

Techniques: Marker

Journal: Physiological Genomics

Article Title: MEF2 transcription factors in human placenta and involvement in cytotrophoblast invasion and differentiation

doi: 10.1152/physiolgenomics.00076.2017

Figure Lengend Snippet: Expression of myocyte enhancer factor-2 (MEF2) family of transcription factors in human extravillous and villous cytotrophoblasts. A: RNA from first-trimester trophoblast cell line (HTR8/SVneo) was analyzed by quantitative (q)RT-PCR for expression of MEF2 isoform transcripts. B: qRT-PCR analysis of MEF2 family members in primary human cytotrophoblasts (CTBs) isolated from term placenta. C: expression of MEF2 isoform mRNA in human choriocarcinoma-derived trophoblastic cell line (BeWo). D: expression of MEF2 family proteins in the indicated human CTB cells. Cell lysates were subjected to Western blot analysis using anti-MEF2A, -2B, -2C, and -2D antibodies. Equal loading was confirmed by stripping and reprobing the same blots with β-actin antibody. E: the histogram indicates average of adjusted MEF2A, 2B, 2C, and 2D levels. Means ± SD, n = 3. **P < 0.001.

Article Snippet: Primers and probes for human MEF2A (Hs01050409_m1), MEF2B (Hs04188747_m1), MEF2C (Hs00231149_m1), MEF2D (Hs00954729_m1), 18S ribosomal RNA (18S rRNA, Hs99999901_sl), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs02758991_g1) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Derivative Assay, Western Blot, Stripping Membranes

Journal: Physiological Genomics

Article Title: MEF2 transcription factors in human placenta and involvement in cytotrophoblast invasion and differentiation

doi: 10.1152/physiolgenomics.00076.2017

Figure Lengend Snippet: Overexpression of MEF2D and dominant negative (dn)MEF2 in human HTR8/SVneo trophoblasts result in enhanced and reduced invasion, respectively. A: representative images of filters containing invaded cells in Matrigel invasion assay. B: invaded cell numbers were determined from 3 independent experiments. Data represent means ± SD (n = 3). * P < 0.05.

Article Snippet: Primers and probes for human MEF2A (Hs01050409_m1), MEF2B (Hs04188747_m1), MEF2C (Hs00231149_m1), MEF2D (Hs00954729_m1), 18S ribosomal RNA (18S rRNA, Hs99999901_sl), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs02758991_g1) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

Techniques: Over Expression, Dominant Negative Mutation, Invasion Assay

Journal: Physiological Genomics

Article Title: MEF2 transcription factors in human placenta and involvement in cytotrophoblast invasion and differentiation

doi: 10.1152/physiolgenomics.00076.2017

Figure Lengend Snippet: Effect of MEF2D and dnMEF2 on extravillous trophoblast cell migration. A: wound healing assay of the HTR8/SVneo cells with overexpression of pcDNA3 control, dnMEF2, or MEF2D. Representative images of cells at 0, 24, and 48 h time point are shown. Images depicting wounds at 0 h are shown at left, whereas images depicting the cell frontiers at 24 and 48 h are shown at middle and right. In each image, the edges of the wound are indicated with vertical light gray lines. B: quantification of migrated cells into wound area. Data represent means ± SD (n = 3). *P < 0.05, **P < 0.001.

Article Snippet: Primers and probes for human MEF2A (Hs01050409_m1), MEF2B (Hs04188747_m1), MEF2C (Hs00231149_m1), MEF2D (Hs00954729_m1), 18S ribosomal RNA (18S rRNA, Hs99999901_sl), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs02758991_g1) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

Techniques: Migration, Wound Healing Assay, Over Expression, Control

Journal: Physiological Genomics

Article Title: MEF2 transcription factors in human placenta and involvement in cytotrophoblast invasion and differentiation

doi: 10.1152/physiolgenomics.00076.2017

Figure Lengend Snippet: Effects of MEF2D, MEF2-VP16, and dnMEF2 on extravillous trophoblast proliferation. HTR8/SVneo cells were transfected with pcDNA3 control, dnMEF2, MEF2D, or MEF2-VP16, respectively, for 24 h. Viabilities of HTR8/SVneo cells were measured by the MTT assay. The experiments were performed in triplicate. Data are presented as means ± SD (n = 3). *P < 0.05.

Article Snippet: Primers and probes for human MEF2A (Hs01050409_m1), MEF2B (Hs04188747_m1), MEF2C (Hs00231149_m1), MEF2D (Hs00954729_m1), 18S ribosomal RNA (18S rRNA, Hs99999901_sl), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs02758991_g1) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA).

Techniques: Transfection, Control, MTT Assay

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: SRC-mediated tyrosine phosphorylation of MEF2D is required for transcriptional activity of MEF2D and in regulation of MTORC1 activity. (A) His-tagged MEF2D- WT or MEF2D mutant constructs (mutation of Tyr33, 57, 69, 72, 117, 131, 225, 333, 337 and 478 residues) were co-transfected with or without Flag-tagged SRC in HEK293T cells for 24 h. Cells were lysed and subjected to immunoprecipitation against His-tag, followed by immunoblotting with p -tyr antibody. (B) HEK293T cells transfected His-tagged MEF2D- WT or MEF2D- 3YF mutants with or without Flag-tagged SRC were lysed and subjected to immunoprecipitation against His-tag, followed by immunoblotting with indicated antibodies. (C) Flag-tagged MEF2D-WT or its 3YF mutant protein purified from HEK293T cells was incubated with commercial active GST-tagged SRC kinase in a kinase assay buffer, followed by immunoblotting with p -tyr antibody. (D) sequence alignment of the residues flanking across different species. Black arrowheads point to the tyrosine residues corresponding to the Tyr333 and tyr 337 residues in human MEF2D. (E) HeLa cells that transfected with Flag-tagged MEF2D- WT or MEF2D- 3YF were maintained in a serum free medium for 4 h, followed with or without EGF treatment. Cell lysates were prepared and immunoprecipitation were analyzed by immunoblotting. (F) luciferase assay was performed in depletion of both MEF2A and MEF2D HeLa cells after co-transfection of indicated expression plasmids and wild-type ( MEF2 reporter-WT) or mutated ( MEF2 reporter-mt) luciferase reporter plasmids for 24 h. (G) qRT-PCR analysis was performed in MEF2A and MEF2D double-knockdown HeLa cells that reconstructed with MEF2D- WT or MEF2D- 3YF. The mRNA levels of FNIP1 , FNIP2 , FLCN , NR4A1 , ZMAT4 and DAAM1 were shown. (H) MEF2A and MEF2D double-knockdown HeLa cells were starved with serum for 4 h and then treated with EGF for 3 h before immunoblotting analysis of the activation of MTORC1 with indicated antibodies. Right plots show phosphorylated p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (bottom) ratios. (I) MEF2A and MEF2D double-knockdown HeLa cells that transfected with indicated plasmids were subjected to serum starvation for 4 h and restimulated with EGF for 3 h. MTORC1 activity was analyzed similarly to (H). Right plots show phosphorylated p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (bottom) ratios. Data are presented as the mean ± S.E.M. (n = 3 independent experiments. two-sided Student’s t-test for H, one-way ANOVA for I, *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phospho-EIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine/p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phospho-SRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Activity Assay, Mutagenesis, Construct, Transfection, Immunoprecipitation, Western Blot, Purification, Incubation, Kinase Assay, Sequencing, Luciferase, Cotransfection, Expressing, Quantitative RT-PCR, Activation Assay

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: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, 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: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, USA).

Techniques: Luciferase, Activity Assay, Control, 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: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, USA).

Techniques: Expressing, Control, 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: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, 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: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, USA).

Techniques: Variant Assay

Journal: Nucleic Acids Research

Article Title: Identification and validation of the pathways and functions regulated by the orphan nuclear receptor, ROR alpha1, in skeletal muscle

doi: 10.1093/nar/gkq180

Figure Lengend Snippet: Relative quantification of gene expression in skeletal muscle of transgenic compared to wt littermate controls

Article Snippet: Mef2d-Mm00504929_m1 , 0.499 , 0.021 , −3.398 , 2.693 , −0.150 , 0.708 , Significant , 0.114 , NS.

Techniques: Quantitative Proteomics, Gene Expression, Transgenic Assay, TaqMan Assay