Journal: Poultry Science

Article Title: METTL14-mediated RNA m 6 A modifications of TET1 and TET2 in duck myoblast differentiation

doi: 10.1016/j.psj.2026.106435

Figure Lengend Snippet: Comparison of RNA m 6 A modification levels in duck leg muscle. (A) DPMs during the proliferation phase; (B) DPMs on day 2 of differentiation (myotubes); (C) Immunofluorescence for Desmin in DPMs during proliferation; (D) Immunofluorescence for MYHC in DPMs on day 4 of differentiation. Dot blot and quantitative analysis of m 6 A in total RNA from duck embryos at E10 and E20 are shown in panels (E) and (F); panels (G) and (H) display quantitative and dot blot analyses of m 6 A in total RNA from DPMs in the GM and DM phases. Methylene blue (MB) staining served as a control for RNA loading. E10 and E20 indicate leg muscle samples from duck embryos at 10 and 20 days of incubation, respectively. GM and DM represent undifferentiated DPMs and DPMs at 48 hours post-differentiation, respectively. * indicates P < 0.05, ** indicates P < 0.01.

Article Snippet: The membrane was blocked in 5% nonfat milk in PBS for 2 hours, followed by overnight incubation at 4°C with primary antibodies against MYHC (DSHB, Iowa, IA, USA; 0.3 μg/mL) and GAPDH (Proteintech, Wuhan, China; 1:50000).

Techniques: Comparison, Modification, Immunofluorescence, Dot Blot, Staining, Control, Incubation

Journal: iScience

Article Title: Histone methyltransferase SET-18/SMYD2-mediated activation of NADase TIR-1d/SARM1 increases mtROS to promote aging

doi: 10.1016/j.isci.2026.114649

Figure Lengend Snippet: SMYD2-mediated activation of SARM1 reduces NAD + level to promote C2C12 myotube atrophy via increase of mtROS (A and B) The effect of smyd2 RNAi in reducing C2C12 myotubes atrophy was abolished by overexpression of SARM1. In A–7F, smyd2 siRNA (siSMYD2) was co-transfected with/without the overexpression plasmid of sarm1 (OE SARM1) into the differentiated C2C12 myotubes. #1 and #2 were two of siRNAs targeting the distinct coding sequences of smyd2 . The non-targeting siRNA (siNT) was used as control. The transfected myotubes were then treated with or without H 2 O 2 , respectively. (A) The representative images of C2C12 myotubes with MHC staining. Red, MHC protein; Blue, the nuclei stained by DAPI. (B) The analysis of myotube diameter and fusion index. The fusion index was indicated as the value of the number of nuclei present in myotubes divided by the number of total nuclei in MHC + cells. Error bars represent SEM. Myotube diameter: without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) = 0.0003, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) < 0.0001, ∗∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) < 0.0001; with H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) < 0.0001, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0365; Fusion index: without H 2 O 2 : ∗p (siSMYD2 #1 vs. siNT) = 0.0162, ∗p (siSMYD2 #2 vs. siNT) = 0.0171, ∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0191, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0101; with H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0017, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0449; one-way ANOVA. (C and D) Overexpression of SARM1 attenuated the influences of smyd2 RNAi in up-regulating MHC mRNA level and downregulating the mRNA levels of atrophy-related genes in C2C12 myotubes with/without H 2 O 2 treatments. The mRNA levels of MHC (C), MuRF1 and Atrogin-1 (D) in myotubes were measured by RT-qPCR and normalized to those of control groups without H 2 O 2 treatment. β-actin was used as an internal reference. Error bars represent SEM. n = 3–4 biological replicates. MHC mRNA level: without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗p (siSMYD2 #2 vs. siNT) = 0.0025, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0003, ∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0025; with H 2 O 2 : ∗∗p (siSMYD2 #1 vs. siNT) = 0.0065, ∗∗p (siSMYD2 #2 vs. siNT) = 0.0069, ∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0246, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0306; MuRF1 mRNA level: without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) = 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0172, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0111; with H 2 O 2 : ∗p (siSMYD2 #1 vs. siNT) = 0.0140, ∗p (siSMYD2 #2 vs. siNT) = 0.0196, ∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0189, ∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0407; Atrogin-1 mRNA level: without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) = 0.0006, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0018, ∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0030; with H 2 O 2 : ∗∗p (siSMYD2 #1 vs. siNT) = 0.0031, ∗∗p (siSMYD2 #2 vs. siNT) = 0.0073, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0002; one-way ANOVA. (E) Overexpression of SARM1 attenuated the increased NAD + level of C2C12 myotubes with smyd2 RNAi treatment. The NAD + levels of myotubes were detected with microplate reader and normalized to those of control without H 2 O 2 treatment. Error bars represent SEM. n = 3 biological replicates. ∗p (siSMYD2 #1 vs. siNT) = 0.0100, ∗p (siSMYD2 #2 vs. siNT) = 0.0451, ∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) = 0.0041, ∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) = 0.0069; one-way ANOVA. (F) Overexpression of SARM1 alleviated the effect of smyd2 RNAi in decreasing mtROS level in C2C12 myotubes with/without H 2 O 2 treatments. The mtROS and nuclei (Blue) in myotubes were stained with MitoSOX and DAPI, respectively. The stained myotubes were detected by confocal microscopy (the representative images showed on the left), followed by quantitative analysis of fluorescence intensity (on the right). Error bars represent SEM. Without H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) < 0.0001, ∗∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) < 0.0001; with H 2 O 2 : ∗∗∗p (siSMYD2 #1 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #2 vs. siNT) < 0.0001, ∗∗∗p (siSMYD2 #1+OE SARM1 vs. siSMYD2 #1) < 0.0001, ∗∗∗p (siSMYD2 #2+OE SARM1 vs. siSMYD2 #2) < 0.0001; one-way ANOVA.

Article Snippet: The C2C12 myotubes were incubated with anti-myosin heavy chain (MHC) antibody (MF20, DSHB) followed by fluorescent secondary antibody (C2181, Sigma Chemical) to identify MHC expression.

Techniques: Activation Assay, Over Expression, Transfection, Plasmid Preparation, Control, Staining, Quantitative RT-PCR, Confocal Microscopy, Fluorescence