Journal: Cytotechnology

Article Title: Novel regulations of MEF2-A, MEF2-D, and CACNA1S in the functional incompetence of adipose-derived mesenchymal stem cells by induced indoxyl sulfate in chronic kidney disease

doi: 10.1007/s10616-016-9983-0

Figure Lengend Snippet: Schematic summary of IS induced ADMSCs functionally incompetence via MEF2A, MEF2D and CACNA1S. The malfunction of ADMSCs subsequently resulted in declined the capacity of stem cells proliferation, migration and wound healing power. Meanwhile, cell apoptosis process has been progressively escalated concomitant with cells blocked at G1 phase. These features eventually hold back the regeneration capacity of chronic kidney disease patients

Article Snippet: Following the manufacturer’s instructions, the primary antibodies (polyclonal antibodies) against phospho-MEF2A (Ser408)(1:1000, Cell Signaling Technology, Danvers, MA, USA), against phosphor-MEF2D (Ser444)(1:1000, GeneTex, Irvine, CA, USA), against CACNA1S (1:1000, GeneTex), and against actin (1:1000, GeneTex) were first incubated with the antigen at a 1 μg: 1 μg ratio at room temperature for 2 h as a pre-absorption control.

Techniques: Migration

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: Cell Death & Disease

Article Title: Long non-coding RNA Irm enhances myogenic differentiation by interacting with MEF2D

doi: 10.1038/s41419-019-1399-2

Figure Lengend Snippet: a RNA-FISH for detecting Irm and GAPDH in undifferentiated and differentiated C2C12 cells. Red: Irm or GAPDH . Blue: DAPI staining. Scale bar, 50 μm. b Relative abundance of Irm in total and nuclear RNAs of differentiating C2C12 cells, as detected by qRT-PCR. U6 , Xist , and GAPDH were used as endogenous controls. c A schematic representation of RNA pull-down assay. d Western blotting assay for the specific interaction of Irm with MEF2D. e A schematic representation of RNA immunoprecipitation (RIP) assay. f RIP assay showed the association of MEF2D with Irm in vivo, as detected by RT-PCR and qRT-PCR. g Deletion mapping of the MEF2D-binding region(s) in Irm . Top: western blotting for MEF2D in protein samples pulled down by the different truncated Irm constructs. Bottom: diagrams of the full-length and truncated Irms . nt: nucleotide. Data shown represent the mean ± SEM of three independent experiments. n.s., not significant; *** P < 0.05 by Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; qRT-PCR, quantitative real-time polymerase chain reaction; RNA-FISH, RNA fluorescence in situ hybridization

Article Snippet: The antibody against MEF2D (Santa Cruz Biotechnology, USA) was used for RIP.

Techniques: Staining, Quantitative RT-PCR, Pull Down Assay, Western Blot, RNA Immunoprecipitation, In Vivo, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Construct, Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization

Journal: Cell Death & Disease

Article Title: Long non-coding RNA Irm enhances myogenic differentiation by interacting with MEF2D

doi: 10.1038/s41419-019-1399-2

Figure Lengend Snippet: a The effect of Irm knockdown on the expression of myogenin and miR-206 depended on MEF2D, which was detected by qRT-PCR. b The effect of Irm knockdown on the differentiation of C2C12 cells depended on MEF2D. Fusion index was calculated. Scale bars, 50 mm. c C2C12 cells were transfected with si-Irm or si-scramble, and the luciferase reporter plasmids were generated by inserting the promoter region of myogenin or miR-206. The luciferase activities were measured 48 h after differentiation. d C2C12 cells were transfected with pc-Irm or pc-Ctrl, and the luciferase reporter plasmids were generated by inserting the promoter region of myogenin or miR-206. The luciferase activities were measured 48 h after differentiation. e Knockdown of Irm impaired the binding ability of MEF2D to myogenin and miR-206 promoters, which was determined by ChIP and qRT-PCR assays. f Knockdown of Irm impaired the binding ability of MyoD to myogenin and miR-206 promoters, which was determined by ChIP and qRT-PCR assays. g Chromatin isolation by RNA purification (ChIRP) assay was performed using even and odd antisense oligos tiling Irm and, a significant amount of genomic DNAs corresponding to myogenin and miR-206 promoters but not in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) locus was retrieved. LacZ ChIRP retrieved no signal. h Model for Irm -regulating myogenesis. Data shown represent the mean ± SEM of three independent experiments. n.s., not significant; * P < 0.05, ** P < 0.05, and *** P < 0.05 by Student’s t test. ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time polymerase chain reaction

Article Snippet: The antibody against MEF2D (Santa Cruz Biotechnology, USA) was used for RIP.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Generated, Binding Assay, Isolation, Purification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: American Journal of Translational Research

Article Title: MiR-665 regulates VSMCs proliferation via targeting FGF9 and MEF2D and modulating activities of Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: FGF9 is a direct target of miR-665. A. The seed sequence of wild type (WT) 3’UTR or mutant (Mut) 3’UTR of FGF9 that is complementary to miR-665; B and C. VSMCs were co-transfected with miR-665 mimics or its control with wild type or mutant 3’UTR of FGF9, and luciferase activity was detected at 48 h post transfection; D. The mRNA and protein expression level of FGF9 in VSMCs was determined by qRT-PCR and western blot, respectively, at 48 h after cells transfected with miR-665 mimics or its control. MiR-665 mimics transfection suppressed the mRNA and protein levels of FGF9. E. The cell proliferation of VSMCs was measured by CKK-8 assay at 48 h after cells co-transfected with miR-665 mimics and pcDNA3.1-FGF9 or their respective controls. Data represents the mean values of three independent replicates ± SEM; significant differences relative to control group were shown as *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The pmirGLO vectors containing wild type or mutant miR-665 binding site in FGF9 3’UTR and MEF2D 3’UTR were synthesized by Ribobio (Guangzhou, China).

Techniques: Sequencing, Mutagenesis, Transfection, Control, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: American Journal of Translational Research

Article Title: MiR-665 regulates VSMCs proliferation via targeting FGF9 and MEF2D and modulating activities of Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: MEF2D is a direct target of miR-665. A. The seed sequence of wild type (WT) 3’UTR or mutant (Mut) 3’UTR of MEF2D that is complementary to miR-665. B and C. VSMCs were co-transfected with miR-665 mimics or its control with wild type or mutant 3’UTR of MEF2D, and luciferase activity was detected at 48 h post transfection. D. The mRNA and protein expression levels of MEF2D in VSMCs was determined by qRT-PCR and western blot, respectively, at 48 h after cells transfected with miR-665 mimics or its control. MiR-665 mimics transfection suppressed the mRNA and protein levels of MEF2D. E. The VSMCs proliferation was measured by CKK-8 assay at 48 h after cells co-transfected with miR-665 mimics and pcDNA3.1-MEF2D or their respective controls. Data represents the mean values of three independent replicates ± SEM; significant differences relative to control group were shown as *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The pmirGLO vectors containing wild type or mutant miR-665 binding site in FGF9 3’UTR and MEF2D 3’UTR were synthesized by Ribobio (Guangzhou, China).

Techniques: Sequencing, Mutagenesis, Transfection, Control, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: American Journal of Translational Research

Article Title: MiR-665 regulates VSMCs proliferation via targeting FGF9 and MEF2D and modulating activities of Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: Schematic illustration demonstrating the role of miR-665 in VSMCs. PDGF-bb or 20% serum treatment suppressed miR-665 expression. MiR-665 inhibited Wnt/β-catenin signaling activities possibly via suppressing FGF9 or MEF2D or by other unidentified mechanism, and the inhibition of FGF9, MEF2D and the activities of Wnt/β-catenin signaling may contribute to the suppression of VSMCs proliferation.

Article Snippet: The pmirGLO vectors containing wild type or mutant miR-665 binding site in FGF9 3’UTR and MEF2D 3’UTR were synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing, Inhibition