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: Cells

Article Title: D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation

doi: 10.3390/cells11071230

Figure Lengend Snippet: Effect of Na 2 S on the amounts of Nrf2 and CMA-related proteins in AD293 cells. ( A ) Immunoblot analyses of Nrf2 and β-actin in cell lysates from AD293 cells treated with Na 2 S (10 μM) for 4 and 24 h. ( B ) Quantitative analyses of Nrf2 amounts from the immunoblot results shown in A. ( C ) Immunoblot analyses of CMA-related proteins (LAMP2A, Hsc70) and β-actin in cell lysates from AD293 cells treated with Na 2 S (10 μM) for 24 h. ( D ) Quantitative analyses of LAMP2A and Hsc70 amounts from the immunoblot results shown in C. ( E ) Immunoblot analyses of a CMA/mA substrate (MEF2D) and β-actin in cell lysates from AD293 cells treated with Na 2 S (10 μM) for 24 h. ( F ) Quantitative analysis of MEF2D amount from the immunoblot results shown in F. Whole blot images are presented in . Amounts of β-actin were used as internal controls for quantification. Numbers in the columns represent the number of samples. * p < 0.05, *** p < 0.001 (unpaired t -test).

Article Snippet: Anti-myocyte enhancer factor 2D (MEF2D) mouse monoclonal antibody (#610774) was obtained from BD Biosciences (San Jose, CA, USA).

Techniques: Western Blot

Journal: Cells

Article Title: D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation

doi: 10.3390/cells11071230

Figure Lengend Snippet: Effect of long-term treatment with D-cysteine on the amounts of Nrf2- and CMA-related proteins in cerebellar lysates from ICR mice. ( A ) Immunoblot analyses of Nrf2, NQO1, LAMP2A, Hsc70, MEF2D, and β-actin in cerebellar lysates from ICR mice daily treated with saline (Sal) and D-cysteine (100 mg/kg/day) for 10 weeks. Whole blot images are presented in . ( B ) Quantitative analyses of the amounts of Nrf2, NQO1, LAMP2A, Hsc70, and MEF2D shown in A. Amounts of β-actin were used as internal controls for the quantification. * p < 0.05, ** p < 0.01 vs. saline-treated mice (unpaired t -test, n = 5 in both saline- and D-cysteine-treated mice).

Article Snippet: Anti-myocyte enhancer factor 2D (MEF2D) mouse monoclonal antibody (#610774) was obtained from BD Biosciences (San Jose, CA, USA).

Techniques: Western Blot

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: The Journal of clinical endocrinology and metabolism

Article Title: Tight glycemic control may increase regenerative potential of myocardium during acute infarction.

doi: 10.1210/jc.2011-2037

Figure Lengend Snippet: FIG. 1. Tight glycemic control increases the number of MPC (c-kitMEFC2GATA4-positive cells) in ischemic myocardium. A, Representative sections show immunohistochemistry for c-kit and MEFC2 colocalization (400), c-kit and GATA4 colocalization (400), and negative control (insets; 400) in serial ischemic myocardial specimens of CGC group, IGC group, GIK infusion group, and normoglycemic group. CPC are shown by the combination of labeling of nuclei by antibody against MEFC2 and GATA4 (red) and staining of myocyte cytoplasm by antibody against c-kit (brown). B, Representative sections show immunofluorescence for c-kit and MEFC2 colocalization, and c-kit and GATA4 colocalization in serial ischemic myocardial specimens of CGC group, IGC group, GIK infusion group, and normoglycemic group. CPC are shown in panel A by the combination of labeling of nuclei by antibody against MEFC2 (insets, green) and GATA4 (insets, green), staining of myocyte cytoplasm by antibody against c-kit (insets, red) and colocalization (yellow). C, Numbers of CPC expressing c-kit, MECF2, and GATA4 in the peri-infarcted area of left ventricle of CGC, ICG, GIK and hearts in the control group, assessed by immunohistochemistry and immunofluorescence analysis. In all box plots, the top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the line in the middle represents the 50th percentile. The whiskers (the lines that extend out the top and bottom of the box) represent the highest and lowest values that are not outliers or extreme values. Outliers (values that are between 1.5 and 3 times the IQR) and extreme values (values that are more than three times the IQR) are represented by circles beyond the whiskers. Double positive cells per field are mean SD of four fields for each patients. *, P 0.05 compared with CGC group; †, P 0.05 compared with ICG group.

Article Snippet: To recognize the MPC, serial sections were incubated with specific antibodies against c-kit (Dako, Glostrup, Denmark), myocyte transcription factors GATA4 and MEF2D (Santa Cruz Biotechnology, Santa Cruz, CA) (21, 22).

Techniques: Control, Immunohistochemistry, Negative Control, Labeling, Staining, Immunofluorescence, Expressing

Journal: Nutrients

Article Title: Dietary Complex and Slow Digestive Carbohydrates Prevent Fat Deposits During Catch-Up Growth in Rats

doi: 10.3390/nu12092568

Figure Lengend Snippet: Markers of muscle performance. ( A ). Creatine kinase activity and ( B ) myocyte enhancer factor 2D (MEF2D) expression as markers of muscle performance and differentiation. NR group, not restricted; RDC group, re-feeding with rapid digestible CHO diet; SDC group, re-feeding with slow digestible CHO diet. * Significant difference with NR group, p < 0.05. # Significant difference with RDC group, p < 0.05.

Article Snippet: Specific antibodies against GLUT4 (Biogenesis Ltd., Poole, UK); total and phospho (Ser473)-Protein Kinase B/Ak strain transforming (PKB/Akt) and Myocyte enhancer factor 2D (MEF2D) (Cell Signaling, Beverly, MA, USA); carbohydrate-responsive element-binding protein (ChREBP), fatty acid synthase (FAS), glucose transporter 1 and 2 (GLUT1 and GLUT2), sterol regulatory element-binding proteins (SREBP) SREBP1 and SREBP2, pyruvate kinase (PKM1/2), pyruvate dehydrogenase kinase (PDK4), ATPase5B, uncoupling proteins (UCP) UCP1 and UCP2 (Santa Cruz Biotechnology (Dallas, TX, USA) were used.

Techniques: Activity Assay, Expressing