c2c12 urine skeletal muscle cells  (ATCC)

Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: NMEVs effectively attenuated palmitic acid-induced senescence in C2C12 cells. (A) Schematic diagram of cell culture and treatment. (B) qRT-PCR analysis of the expression of senescence markers p53, cdkn1a, and cdkn2a in each group (n = 3). (C-C‴) Western blotting for the expression of senescence markers p53, cdkn1a (p21), and cdkn2a (p16) in each group with relative quantification (n = 6). (D-D′) Immunofluorescence staining for the DNA damage marker γH2AX with quantitative analysis (Scale bar, 100 μm; n = 6 for each group). (E-E′) Immunofluorescence staining for p16 with quantitative analysis (Scale bar, 100 μm; n = 6). (F-F′) Immunofluorescence staining for p21 with quantitative analysis (Scale bar, 100 μm; n = 6). (G-G′) Flow cytometry analysis of relative reactive oxygen species (ROS) levels (n = 6). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant. Rel. fold, relative fold; T.Ar, total area.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, Quantitative Proteomics, Immunofluorescence, Staining, Marker, Flow Cytometry

Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: NMEVs alleviated palmitic acid-induced mitochondrial dysfunction and lipid deposition. (A) Relative ATP synthesis rates in each group (n = 6). (B) qRT-PCR analysis of MT-CO1 expression in each group (n = 3). (C) qRT-PCR analysis of MT-ND1 expression in each group (n = 3). (D) qRT-PCR analysis of MT-CO3 expression in each group (n = 3). (E) qRT-PCR analysis of D-loop expression in each group (n = 3). (F) Mitochondrial complex V activity in C2C12 cells of each group (n = 6). (G) Measurement of oxygen consumption rate (OCR) in C2C12 cells of each group (n = 4). (H-H′) Transmission electron microscopy (TEM) assessment of mitochondrial quantity with quantitative analysis (Scale bar, 500 nm; n = 3). (I-I′) Western blotting for PGC-1α expression in each group with relative quantification (n = 6). (J-J′) Immunofluorescence staining for SDHA with quantitative analysis (Scale bar, 100 μm; n = 6). (K-K′) Immunofluorescence staining for EdU with quantitative analysis (Scale bar, 100 μm; n = 6). (L-L′) Representative images of BODIPY staining in each group with quantitative analysis (Scale bar, 20 μm; magnified Scale bar, 5 μm; n = 6). (M-M′) Representative images of Oil Red O staining in each group with quantitative analysis (Scale bar, 20 μm; magnified scale bar, 5 μm; n = 6). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Activity Assay, Transmission Assay, Electron Microscopy, Western Blot, Quantitative Proteomics, Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: NMEVs reduced C2C12 senescence and lipid accumulation by enriching miR-542-3p to stabilize mitochondrial function. (A) PCA plot showing sample homogeneity of AMEVs and NMEVs (n = 3). (B) Heatmap showing the top 20 significantly upregulated and downregulated microRNAs. (C) qRT-PCR analysis of the expression of the top 12 significantly upregulated microRNAs (n = 3). (D) qRT-PCR analysis of miR-542-3p expression in C2C12 cells after transfection with NMEVs, mimic, or NMEVs + inhibitor (n = 3). (E-E′) Immunofluorescence staining for p16 with quantitative analysis (Scale bar, 50 μm; n = 6). (F-F′) Immunofluorescence staining for p21 with quantitative analysis (Scale bar, 50 μm; n = 6). (G-G′) Immunofluorescence staining for γH2AX with quantitative analysis (Scale bar, 50 μm; n = 6). (H-H′) Immunofluorescence staining for EdU with quantitative analysis (Scale bar, 50 μm; n = 6). (I-I′) Immunofluorescence staining for SDHA with quantitative analysis (Scale bar, 50 μm; n = 6). (J-J‴) Western blotting for p16, p21 and PGC-1α expression in each group with relative quantification (n = 6). (K) Relative ATP synthesis rates in each group (n = 6). (L) qRT-PCR analysis of MT-CO1 expression in each group (n = 3). (M) qRT-PCR analysis of MT-ND1 expression in each group (n = 3). (N) qRT-PCR analysis of MT-CO3 expression in each group (n = 3). (O) qRT-PCR analysis of D-loop expression in each group (n = 3). (P-P′) Representative images of BODIPY staining in each group with quantitative analysis (Scale bar, 20 μm; magnified Scale bar, 10 μm; n = 6). (Q-Q′) Representative images of Oil Red O staining in each group with quantitative analysis (Scale bar, 20 μm; magnified Scale bar, 10 μm; n = 6). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Immunofluorescence, Staining, Western Blot, Quantitative Proteomics

Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: Asxl2 and Eef1a1 served as downstream target genes of miR-542-3p. (A) Prediction of downstream target genes of miR-542-3p using multiple target gene prediction software. (B) qRT-PCR analysis of Asxl2 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (C) qRT-PCR analysis of Eef1a1 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (D) qRT-PCR analysis of Lrrc59 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (E) qRT-PCR analysis of Gabarap expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (F) qRT-PCR analysis of Ap3d1 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (G) qRT-PCR analysis of Arhgap5 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (H) qRT-PCR analysis of Kcmf1 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (I) qRT-PCR analysis of Pten expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (J) qRT-PCR analysis of Ube2e1 expression in C2C12 cells after transfection with miR-542-3p mimic (n = 3). (K-K″) Western blotting for Asxl2 and Eef1a1 expression in C2C12 cells after transfection with miR-542-3p mimic, with relative quantification (n = 3). (L-L′) Dual-luciferase reporter assay verifying the direct targeting binding relationship between miR-542-3p and Asxl2 (n = 3). (M-M′) Dual-luciferase reporter assay verifying the direct targeting binding relationship between miR-542-3p and Eef1a1 (n = 3). (N-N″) Western blotting for Asxl2 and Eef1a1 expression in neonatal and aging muscle tissues (n = 3). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Software, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Quantitative Proteomics, Luciferase, Reporter Assay, Binding Assay

Journal: Bioactive Materials

Article Title: Neonatal muscle-derived extracellular vesicles containing miR-542-3p rejuvenate aged skeletal muscle via a functional microneedle patch

doi: 10.1016/j.bioactmat.2026.06.011

Figure Lengend Snippet: miR-542-3p suppressed Eef1a1 to ameliorate PA-induced mitochondrial dysfunction and cellular senescence. (A-A′) Western blotting for Eef1a1 expression after PA induction, followed by transfection with miR-542-3p mimic and Eef1a1 overexpression plasmid (Eef1a1 OE ), with relative quantification (n = 3). (B) Schematic diagram illustrating Eef1a1 regulation of lipid storage via AMPK. (C-C′) Western blotting for AMPK and p-AMPK expression in neonatal and aging muscle tissues with relative quantification (n = 3). (D-D′) Western blotting for AMPK and p-AMPK expression after PA induction, followed by transfection with miR-542-3p mimic and Eef1a1 overexpression plasmid (Eef1a1 OE ), with relative quantification (n = 3). (E-E″) Representative images of p16 and p21 staining in each group with quantitative analysis (Scale bar, 50 μm; n = 6). (F) Relative ATP synthesis rates in each group (n = 6). (G) qRT-PCR analysis of MT-CO1 expression in each group (n = 3). (H) qRT-PCR analysis of MT-ND1 expression in each group (n = 3). (I) qRT-PCR analysis of MT-CO3 expression in each group (n = 3). (J) qRT-PCR analysis of D-loop expression in each group (n = 3). (K) Mitochondrial complex V activity in C2C12 cells of each group (n = 6). (L-L′) Representative images of SDHA staining in each group with quantitative analysis (Scale bar, 50 μm; n = 6). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: The C2C12 cell line was procured from Procell Life Science & Technology Co., Ltd (Wuhan, China).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative Proteomics, Staining, Quantitative RT-PCR, Activity Assay