Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: RMS survival is selectively regulated by MyoD (A) Representative images of immunohistochemistry staining of MyoD and myogenin in patient embryonal and alveolar tumors. (B) Histologic images in (A) were scored for MyoD and myogenin immunohistochemistry staining positivity, n = 5. (C) RH30-SR cells expressing a scrambled-control sh-RNA or sh-RNA targeting myogenin were treated with TNF and DOX for 24 h and subsequently measured for cell death, n = 3. (D) RNA was prepared from cells in (C) and qPCR analysis was performed probing for myogenin and its target genes, n = 3. (E) RH30-SR cells expressing a scrambled-control sh-RNA or sh-RNA against either MyoD, MYF5, myogenin, MRF4, MEF2C, and MEF2D (sh-MRFs) were treated with TNF for 24 h and cell death was subsequently measured, n = 3. (F) CHRONOS Score for MRFs in sarcoma cell lines tested in DepMap. RMS cell lines are highlighted in purple, with a score of < −1 indicating a gene that is essential; ATRT, atypical teratoid rhabdoid tumors; ES, Ewing sarcoma; FS, follicular sarcoma; LMS, leiomyosarcoma; LS, liposarcoma; MRT, malignant rhabdoid tumor; PS, pleomorphic sarcoma; RMS, rhabdomyosarcoma; SS, synovial sarcoma; TS, thyroid sarcoma; US, undifferentiated sarcoma. (G and H) Kaplan-Meier curve, log rank test, showing the correlation of both (G) MyoD and (H) myogenin expression stratified by median patient expression to overall survival of rhabdomyosarcoma patients from an R2 Genomics Analysis and Visualization Platform. (I) CHRONOS Score for p65 in sarcoma cell lines tested in the Cancer Dependency Map. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01. See also .

Article Snippet: Antibodies used in this study included, IκBα (Cell Signaling Technology Cat# 9242, RRID: AB_331623 ), α-tubulin (Thermo Fisher Scientific Cat# A11126, RRID: AB_2534135 ), p65/RelA (Santa Cruz Biotechnology Cat# sc-109, RRID: AB_632039 ), MyoD (Santa Cruz Biotechnology Cat# sc-377460, RRID: AB_2813894 ), Myf5 (Santa Cruz Biotechnology Cat# sc-518039), myogenin (Santa Cruz Biotechnology Cat# sc-52903, RRID: AB_784707 ), MRF4 (Santa Cruz Biotechnology sc-514379), MEF2C (Abcam Cat# ab211493, RRID: AB_2864417 ), MEF2D (Santa Cruz Biotechnology Cat# sc-271153, RRID: AB_10614669 ), MYHC (Sigma-Aldrich Cat# M8421, RRID: AB_477248 ), TnnT (Sigma-Aldrich Cat# SAB4200717, RRID: AB_2892079 ), CYLD (Cell Signaling Technology Cat# 4495, RRID: AB_10557111 ), p -RIPK1 (Proteintech Cat# 66854-1-Ig, RRID: AB_2882194 ), RIPK1 (Thermo Fisher Scientific Cat# PA5-20811, RRID: AB_11154790 ), DNMT1 (Novus Cat# NB100-56519, RRID: AB_838131 ), DNMT3A (Santa Cruz Biotechnology Cat# sc-365769, RRID: AB_10844010 ), DNMT3B (Cell Signaling Technology Cat# 67259, RRID: AB_2799723 ), and HRP conjugated secondary antibodies anti-mouse IgG (Promega Cat# W4021, RRID: AB_430834 ) and anti-rabbit IgG (Promega Cat# W4011, RRID: AB_430833 ).

Techniques: Immunohistochemistry, Staining, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Transcription factor MEF2D regulates aberrant expression of ACSL3 and enhances sorafenib resistance by inhibiting ferroptosis in HCC

doi: 10.3389/fphar.2024.1464852

Figure Lengend Snippet: The transcription factor MEF2D regulates aberrant expression of ACSL3 (A) GSEA revealed enrichment of the peroxisome pathway in MEF2D-silenced HCC cells. (B) mRNA expression levels of ACSL3 and other oxidative stress-related candidate genes were analyzed in lv-shMEF2D-infected HCC cells and compared with controls (C) In PLC/PRF/5 cells infected with Lv-MEF2D or Lv-GFP, the mRNA expression of ACSL3 was quantitatively detected by qPCR, and the protein expression level was detected by Western blotting. Vinculin protein was used as an internal control. (D) Bioinformatic analysis for putative MEF2D-binding sites in the regulatory regions of ACSL3 was shown (E) Luciferase expression driven by ACSL3 promoter regions was measured in PLC/PRF/5 and Huh7 cells infected with Lv-MEF2D or Lv-shMEF2D. The folding changes of luciferasee relative activity in Lv-MEF2D- and Lv-shMEF2D infected cells were normalized to Lv-GFP- and Lv-scramble- infected cells, respectively. The data represent the mean ± SDs of three independent experiments. (F) A ChIP assay was performed to detect the binding of MEF2D to the potential MRE identified in the promoter regions of ACSL3. The IgG-incubated and blank groups were considered as negative controls, whereas the input fraction was the positive control (G) The mRNA expression levels of MEF2D in HCC tissues and normal tissues were analyzed using data from the TCGA database (H) The correlation between ACSL3 and MEF2D expression in HCC was analyzed using data from the TCGA database. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Cell lysates were pre-clarified with protein A/G beads and then incubated overnight at 4°C with protein A/G beads coated with anti-MEF2D antibody (2 μg, Santa cruz).

Techniques: Expressing, Infection, Western Blot, Control, Binding Assay, Luciferase, Activity Assay, Incubation, Positive Control

Journal: Journal of Advanced Research

Article Title: Tetramethylpyrazine nitrone exerts neuroprotection via activation of PGC-1α/Nrf2 pathway in Parkinson’s disease models

doi: 10.1016/j.jare.2023.11.021

Figure Lengend Snippet: Information about primary antibodies used.

Article Snippet: MEF2D , Mouse monoclonal , IgG3 , WB, 1:500 IF, 1:200 , sc-271153 , Santa Cruz.

Techniques:

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