Journal: Frontiers in Veterinary Science

Article Title: MEF2C Expression Is Regulated by the Post-transcriptional Activation of the METTL3-m 6 A-YTHDF1 Axis in Myoblast Differentiation

doi: 10.3389/fvets.2022.900924

Figure Lengend Snippet: The protein expression of MEF2C is regulated by m6A modification. (A,B) MEF2C and FTO protein levels after FTO knockdown and overexpression on the 4th day after differentiation. (C,D) MEF2C and METTL3 protein levels after METTL3 knockdown and overexpression for 4th day post differentiation. (E,F) Treatment with a global methylation inhibitor, 3-Deazaadenosine (DAA), for 24 and 48 h led to the downregulation of the MEF2C protein levels in bovine skeletal myoblasts. The results are presented as the means ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, siNC vs. siRNA samples and DMSO vs. DAA at 24h; # p < 0.05, # # # p < 0.001, empty vector vs. overexpression plasmid samples and DMSO vs. DAA at 48h), using Student's t test.

Article Snippet: Additionally, MEF2C protein level was substantially decreased after 24 and 48 h of treatment with a global methylation inhibitor, 3-deazaadenosine (DAA) (4 μM, 86583-19-9, TargetMOI, MA, USA), without affecting its mRNA expression ( , ).

Techniques: Expressing, Modification, Knockdown, Over Expression, Methylation, Plasmid Preparation

Journal: Frontiers in Veterinary Science

Article Title: MEF2C Expression Is Regulated by the Post-transcriptional Activation of the METTL3-m 6 A-YTHDF1 Axis in Myoblast Differentiation

doi: 10.3389/fvets.2022.900924

Figure Lengend Snippet: MEF2C promotes the differentiation of bovine myoblasts by posttranscriptional activation of the METTL3-m 6 A-YTHDF1 axis. METTL3 catalyzes the N 6 -methylation of MEF2C mRNA, and YTHDF1 recognizes this modification site to promote the translation of MEF2C. Then, MEF2C can directly bind to the promoter region of METTL3 to activate its expression, suggesting that there is a positive feedback loop underlying the process of bovine skeletal myoblast differentiation.

Article Snippet: Additionally, MEF2C protein level was substantially decreased after 24 and 48 h of treatment with a global methylation inhibitor, 3-deazaadenosine (DAA) (4 μM, 86583-19-9, TargetMOI, MA, USA), without affecting its mRNA expression ( , ).

Techniques: Activation Assay, Methylation, Modification, Expressing

Journal: Oncotarget

Article Title: Synergistic antitumor effect of a γ-secretase inhibitor PF-03084014 and sorafenib in hepatocellular carcinoma

doi: 10.18632/oncotarget.26209

Figure Lengend Snippet: ( A ) HCC 97H-derived spheroid CSCs were treated with vehicle (DMSO) and sorafenib (sor, 1, 3, 5 μM (left panel) or PF-03084014 (0.1, 0.25 μM) (middle panel) alone, or PF-03084014 0.1 μM in combination with sorafenib 1, 3, and 5 μM, respectively (right panel). Overall spheroid formation (treatment vs. con) was calculated as a percentile. The data are presented as the mean ± SD, n = 3 from three independent experiments, each in triplicate. An independent t test was used for statistical comparison. * p < 0.05; ** p < 0.01; *** p < 0.001. A synergistic effect was considered as the inhibition effect by PF-03084014 + sorafenib was greater than the sum of the effect by PF-03084014 and sorafenib alone. ( B ) 97H spheroids were pre-treated with PF-03084014 for 24 hrs followed by the addition of sorafenib (GSI > sorafenib). ( C ) The single spheroid formation capacity in the control was defined as 1, and the fold decrease in the PF-03084014 (0.1 μM) sorafenib (1 μM), or PF-03084014 + sorafenib groups were calculated as the inverse of the fold change. ( D ) Phase contrast images of spheroid colonies after treatment with DMSO or PF-03084014 and sorafenib alone, or PF-03084014 in combination with sorafenib.

Article Snippet: Sorafenib (Selleck Chemicals) (1–5 μM), γ-secretase inhibitor PF-03084014 (Pfizer Global Research and Development) (0.1–0.25 μM), and the combination of the two drugs were administrated 1–2 times at the indicated dosages.

Techniques: Derivative Assay, Inhibition

Journal: Oncotarget

Article Title: Synergistic antitumor effect of a γ-secretase inhibitor PF-03084014 and sorafenib in hepatocellular carcinoma

doi: 10.18632/oncotarget.26209

Figure Lengend Snippet: ( A ) Schema of the experimental setup. The 97H spheroid-generated subcutaneous tumor cubes were implanted into the left liver lobes of nude mice. One week after tumor implantation, the mice were randomized and treated orally with vehicle, PF-03084014, sorafenib, and PF-03084014 + sorafenib. ( B ) Tumor growth, based upon the luciferin bioluminescence signal, at 2 and 4 weeks. Data are presented as the mean ± SD, mouse number = 7 in each group. ** p < 0.01. ( C ) Representative tumor bioluminescence images at 2 and 4 weeks in vehicle, PF-03084014, sorafenib, and PF-03084014 + sorafenib, respectively. ( D ) Statistical comparison of the tumor volumes measured at the end point of the study (4 weeks). * p < 0.05. ( E ) Orthotopic tumor incidence (%) of the respective treatment groups at the end point.

Article Snippet: Sorafenib (Selleck Chemicals) (1–5 μM), γ-secretase inhibitor PF-03084014 (Pfizer Global Research and Development) (0.1–0.25 μM), and the combination of the two drugs were administrated 1–2 times at the indicated dosages.

Techniques: Generated, Tumor Implantation

Journal: Oncotarget

Article Title: Synergistic antitumor effect of a γ-secretase inhibitor PF-03084014 and sorafenib in hepatocellular carcinoma

doi: 10.18632/oncotarget.26209

Figure Lengend Snippet: ( A ) HCC spheroids treated with vehicle, PF-03084014, sorafenib, and PF-03084014 + sorafenib for 48–72 hrs followed by qRT-PCR analysis. The statistical comparison is of mRNA levels of SNAIL1, SNAIL2, CDH2, VIMENTIN, and CDH1 in drug treated cells versus control. ( B ) mRNA levels of stemness genes NANOG and OCT4 in drug treated cells versus control. ( C ) mRNA levels of ABCG2 and ABCB1. qPCR data are the mean ± SD, n = 2. * p < 0.05; ** p < 0.01; *** p < 0.001. ( D ) At the end of study, tumors were harvested from mice as described in Materials and Methods. Proteins were extracted and subjected to immunoblot analysis for phospho-Erk and phospho-Akt. ( E ) Immunoblot analysis for E-cadherin and Snail1. ( F ) Immunoblot for stemness proteins.

Article Snippet: Sorafenib (Selleck Chemicals) (1–5 μM), γ-secretase inhibitor PF-03084014 (Pfizer Global Research and Development) (0.1–0.25 μM), and the combination of the two drugs were administrated 1–2 times at the indicated dosages.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Frontiers in Veterinary Science

Article Title: MEF2C Expression Is Regulated by the Post-transcriptional Activation of the METTL3-m 6 A-YTHDF1 Axis in Myoblast Differentiation

doi: 10.3389/fvets.2022.900924

Figure Lengend Snippet: The protein expression of MEF2C is regulated by m6A modification. (A,B) MEF2C and FTO protein levels after FTO knockdown and overexpression on the 4th day after differentiation. (C,D) MEF2C and METTL3 protein levels after METTL3 knockdown and overexpression for 4th day post differentiation. (E,F) Treatment with a global methylation inhibitor, 3-Deazaadenosine (DAA), for 24 and 48 h led to the downregulation of the MEF2C protein levels in bovine skeletal myoblasts. The results are presented as the means ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, siNC vs. siRNA samples and DMSO vs. DAA at 24h; # p < 0.05, # # # p < 0.001, empty vector vs. overexpression plasmid samples and DMSO vs. DAA at 48h), using Student's t test.

Article Snippet: Additionally, MEF2C protein level was substantially decreased after 24 and 48 h of treatment with a global methylation inhibitor, 3-deazaadenosine (DAA) (4 μM, 86583-19-9, TargetMOI, MA, USA), without affecting its mRNA expression ( , ).

Techniques: Expressing, Modification, Over Expression, Methylation, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Arginine methylation of the C-terminus RGG motif promotes TOP3B topoisomerase activity and stress granule localization

doi: 10.1093/nar/gky103

Figure Lengend Snippet: TOP3B is methylated in cells. ( A ) Endogenous TOP3B was immunoprecipitated from control and Adox-treated (20 μM, 48 h) HeLa cells under denaturing conditions. TOP3B methylation was detected using a pan-anti-ADMA antibody. Immunoprecipitated TOP3B was detected with an anti-TOP3B antibody. ( B ) An immunoprecipitation assay was performed using an anti-ADMA antibody to enrich arginine-methylated proteins from control and Adox treated HeLa cells. The immunoprecipitated protein complexes were detected with anti-TOP3B antibody. The expression of TOP3B was detected in input cell lysates. ( C ) Endogenous TOP3B was immunoprecipitated from control and MS023-treated (10 μM, 48 h) HeLa cells under denaturing conditions. TOP3B methylation was detected using an anti-ADMA antibody, as described in (A). Immunoprecipitated TOP3B was detected with an anti-TOP3B antibody. ( D ) Endogenous TOP3B was immunoprecipitated from control, Adox and MS023-treated HeLa cells under denaturing conditions. TOP3B methylation was detected using a pan-anti-MMA antibody. Immunoprecipitated TOP3B was detected with an anti-TOP3B antibody. ( E ) HeLa cells were transfected with Flag-TOP3B and Flag-TOP3B (R833/835K) constructs for 48 h. The total cell lysates were prepared under denaturing conditions and immunoprecipitated with an anti-FLAG antibody. The immunoprecipitated protein samples were detected with anti-ADMA and anti-FLAG antibodies. In panels A–E, the input samples were detected with either anti-ADMA or anti-MMA antibody to examine the global methylation level of the input cell lysates.

Article Snippet: The global methylation inhibitor adenosine dialdehyde (AdOx) (A7154) was purchased from Sigma.

Techniques: Methylation, Immunoprecipitation, Expressing, Transfection, Construct