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: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 5. Dex reversed H/R-induced upre gulation of miR-665 and downregulation of MEF2D. (A-B) Expressions of miR-665 and MEF2D mRNA were examined by qRT-PCR. (C-D) Expressions of MEF2D protein were detected by Western blot. Quantitative analyses of protein band intensity. GAPDH served as an internal control for sample loading. (E) Predicted duplex formation between MEF2D 3′- UTR and miR-665. (F) Dual luciferase gene reporter assay manifested that miR- 665 could directly bind with MEF2D. (n = 3 per group). **p < 0.01, ***p < 0.001, ****p < 0.0001. qRT- PCR, quantitative reverse transcription PCR; UTR, untranslated region; MEF2D, myocyte enhancer factor 2D.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Quantitative RT-PCR, Western Blot, Control, Luciferase, Reporter Assay, Reverse Transcription

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 6. Dex improved cell viability and decreased apoptosis of H9c2 cells undergoing H/R via downregulation of miR-665 followed by upregulation of MEF2D. The H9c2 cells were transfected with miR-665mimics or co-transfection of miR-665mimics with pcDNA-MEF2D for 48 h before treatment with 10 nM Dex for 1 h. (A) The effects of miR-665 overexpression or miR-665 and MEF2D simultaneous overexpression on morphology of H9c2 cells in each group were observed using an inverted microscope (scale bars, 100 µm). (B) The effects of miR-665 overexpression or miR-665 and MEF2D simultaneous overexpression on the cell viability were detected using CCK-8 assay. (C-F) The effects of miR-665 overexpression or miR-665 and MEF2D simultaneous overexpression on the cell apoptosis were gauged by flow cytometry. The apoptotic rates were presented as addition of the percentages of cells at early apoptotic phase and late apoptotic phase. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Transfection, Cotransfection, Over Expression, Inverted Microscopy, CCK-8 Assay, Flow Cytometry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 7. Dex protected against pyroptosis of H9c2 cells undergoing H/R via downregulation of miR-665 followed by upregulation of MEF2D. (A) Protein bands of MEF2D, IL-1β, IL-18, NLRP3, ASC, C-Caspase-1, and GSDMD were evaluated by Western blot. (B-C) Expressions of miR-665 and MEF2D mRNA were detected by qRT- PCR. (D-J) Quantitative analyses of protein band intensities of MEF2D, IL-1β, IL-18, NLRP3, ASC, C-Caspase-1, and GSDMD. GAPDH served as an internal control for sample loading. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Western Blot, Quantitative RT-PCR, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 8. Dex facilitated nuclear translocation of Nrf2 regulated by MEF2D in H/R-treated H9c2 cells. (A) Expressions of cytoplasmic Nrf2 and nuclear Nrf2 proteins were determined by Western blot in H/R-treated H9c2 cells subjected to Dex pretreatment. GAPDH and Lamin B were used as internal references for sample loading respectively. (B-C) Quantitative analyses of the expression levels of cytoplasmic Nrf2 and nuclear Nrf2. (D) Expressions of cytoplasmic Nrf2 and nuclear Nrf2 proteins were detected by Western blot in H/R-treated H9c2 cells undergoing Dex pretreatment and transfection of miR-665mimics or co-transfection of miR-665mimics with pcDNA-MEF2D respectively. GAPDH and Histone3 acted as internal references for sample loading respectively. (E-F) Quantitative analyses of the expression levels of cytoplasmic Nrf2 and nuclear Nrf2. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant. Nrf2, nuclear factor erythroid 2-related factor 2.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Translocation Assay, Western Blot, Expressing, Transfection, Cotransfection

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Human muscle-specific A-kinase anchoring protein polymorphisms modulate the susceptibility to cardiovascular diseases by altering cAMP/PKA signaling

doi: 10.1152/ajpheart.00034.2018

Figure Lengend Snippet: There was no change in the expression of calcineurin and phospho-MEF2D before or after isoproterenol (ISO) in mutant muscle-specific A-kinase anchoring protein-expressing human embryonic kidney-293T cells. A: representative immunoblots of bands obtained from calcineurin and GAPDH antibodies and the corresponding quantification bar graph. Values are means ± SE obtained from four independent experiments [control (−): 0.9312 ± 0.04733; control (+): 0.9992 ± 0.04312; wild-type (WT) (−): 1.019 ± 0.005362; WT (+): 1.032 ± 0.01538; S1653R (−): 1.018 ± 0.06504; S1653R (+): 1.185 ± 0.08820; E2124G (−): 1.064 ± 0.01953; E2124G (+): 1.054 ± 0.05452]. B: representative Western blot image of phospho-MEF2D (Ser444) and total MEF2D with respective bar graph quantification. Four separate experiments were performed for analysis. Values are expressed as means ± SE [control (−): 1.009 ± 0.02920; control (+): 1.065 ± 0.08603; WT (−): 0.9977 ± 0.04492; WT (+): 1.010 ± 0.06008; S1653R (−): 1.065 ± 0.06702; S1653R (+): 1.116 ± 0.06349; E2124G (−): 1.064 ± 0.06982; E2124G (+): 1.078 ± 0.08712]. −, No stimulation or absence of stimulation; +ISO stimulation, where indicated. IB, immunoblot analysis.

Article Snippet: Primary antibodies with the following dilutions and manufacturers were used: mAKAP (1:2,000, BioLegend), PKA RIIα (1:500, Santa Cruz Bioetchnology), PKA-Cα (1:1,000, Cell Signaling Technology), PP2A C-subunit (1:1,000, Cell Signaling Technology), PDE4D3 (1:500, Abcam), GAPDH (1:10,000, Cell Signaling Technology), cAMP response element-binding protein (CREB) and phospho-CREB (Ser 133 ) (1:1,000, Cell Signaling Technology), calcineurin (1:1,000, Cell Signaling Technology), histone deacetylase 4 (HDAC4) and phospho-HDAC4 (Ser 467/632 ) (1:1,000, Cell Signaling Technology), PKD (1:500, Abcam), phospho-PKD (Ser 916 ) (1:500, Cell Signaling Technology), myocyte-specific enhancer factor 2D (MEF2D; 1:500, GeneTex), phospho-MEF2D (1:500, GeneTex), β 1 -adrenergic receptor (1:500, Abcam), and β 2 -adrenergic receptor (1:500, Abcam).

Techniques: Expressing, Mutagenesis, Western Blot

Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 stimulates MEF2D/PGC1α/Nrf2 signal pathway through regulation of the Akt/GSK3β pathway in PD animal models. ( A ) Representative Western blots and densitometric analysis the expression of CDK5, p-MEF2D, MEF2D and PGC1α. ( B , E ) Representative Western blots and densitometric analysis of the expression of p-Akt and p-GSK3β. ( C , F ) Representative Western blots and densitometric analysis of the expression of Nrf2, HO-1 and TFAM. ( D ) Representative Western blots and densitometric analysis the expression of MEF2D and PGC1α. Data are expressed as mean±SEM (n=3 per group). # P<0.05, ## P<0.01, ### P<0.001 vs. sham group, and * P<0.05, ** P<0.01 and *** P<0.001vs. MPTP or 6-OHDA group.

Article Snippet: After blocking with a blocking buffer, the polyvinyldifluoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TH (Cell Signaling Technology), CDK5, phospho-Ser444-MEF2D (p-MEF2D, Merck Millipore, Germany), MEF2D (Merck Millipore, Germany), PGC1α, Nrf2, HO-1, TFAM, phospho-Ser473-Akt (p-Akt), total-Akt (Akt), p-GSK3β, total-GSK3β (GSK3β), phospho-Ser133-CREB (p-CREB, Cell Signaling Technology), CREB (Cell Signaling Technology), mature BDNF (abcam, Britain), synaptophysin (SYP, Cell Signaling Technology) and β-actin.

Techniques: Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 prevents the loss of SN DA neuron loss by activating MEF2D/PGC1α/GSK3β signal pathway. Representative images of middle brain sections co-stained with antibodies against ( A , D ) TH (green) and p-GSK3β (red); ( B , E ) TH (red) and MEF2D (green); ( C , F ) TH (green) and PGC1α (red). DAPI (blue) indicates nucleus. ( G , H ) Quantitative analysis of immunofluorescence intensity in TH-positive cells. Data are expressed as mean±SEM (n=3 to 4 per group). # P<0.05, ### P<0.001 vs. sham group, and * P<0.05, ** P<0.01 vs. MPP + or 6-OHDA group.

Article Snippet: After blocking with a blocking buffer, the polyvinyldifluoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TH (Cell Signaling Technology), CDK5, phospho-Ser444-MEF2D (p-MEF2D, Merck Millipore, Germany), MEF2D (Merck Millipore, Germany), PGC1α, Nrf2, HO-1, TFAM, phospho-Ser473-Akt (p-Akt), total-Akt (Akt), p-GSK3β, total-GSK3β (GSK3β), phospho-Ser133-CREB (p-CREB, Cell Signaling Technology), CREB (Cell Signaling Technology), mature BDNF (abcam, Britain), synaptophysin (SYP, Cell Signaling Technology) and β-actin.

Techniques: Staining, Immunofluorescence

Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 activates the MEF2/PGC1α/Nrf2 pathway through regulation of the Akt-GSK3β pathway. A53T and corrected DA neurons treated with T-006 and the positive drug at the indicated concentration for 24 hours. For MPP + -treatment assay, CGNs were pretreated with or without LY294002 (1 μM), Akt-iv (1 μM), or LiCl (10 μM) for 2 h, incubated with or without T-006 for 2 h, and finally exposed to MPP + . Cell viability was examined using an MTT assay. Luciferase reporter gene assays respectively included MEF2 ( A ), PGC1α ( B ), PGC1α-ΔMEF2 ( C ) and ARE ( D ). ( E ) Representative images of neurons co-stained with antibody against Nrf2 (green). DAPI (blue) indicates nucleus. ( F ) Luciferase reporter gene assays of MEF2 with MPP + induction. ( G – K ) respectively represent the fold changes of CDK5, MEF2D, PGC1α, Nrf1 and Nrf2 at mRNA level. ( L ) Effect of MEF2D reduction on MPP + -induced neurotoxicity in CGNs. ( M ) Effects of Akt pathway inhibitor LY294002 and GSK3β inhibitor on MEF2 transcriptional activity. ( N ) Effects of Akt pathway inhibitors LY294002 and Akt-iv, and GSK3β inhibitor on MPP + -induced neurotoxicity in CGNs. Data above are all from three independent experiments, expressed as mean±SEM. # P<0.05, ## P<0.01 and ### P<0.001 vs. control (Ctrl) group, and * P<0.05, ** P<0.01, *** P<0.001 vs. MPP + group.

Article Snippet: After blocking with a blocking buffer, the polyvinyldifluoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TH (Cell Signaling Technology), CDK5, phospho-Ser444-MEF2D (p-MEF2D, Merck Millipore, Germany), MEF2D (Merck Millipore, Germany), PGC1α, Nrf2, HO-1, TFAM, phospho-Ser473-Akt (p-Akt), total-Akt (Akt), p-GSK3β, total-GSK3β (GSK3β), phospho-Ser133-CREB (p-CREB, Cell Signaling Technology), CREB (Cell Signaling Technology), mature BDNF (abcam, Britain), synaptophysin (SYP, Cell Signaling Technology) and β-actin.

Techniques: Concentration Assay, Incubation, MTT Assay, Luciferase, Staining, Activity Assay, Control