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

Journal: Molecular Therapy. Nucleic Acids

Article Title: Single-base 2′OMe-modified LNA and MOE gapmers selectively silence ACVR1 R206H in fibrodysplasia ossificans progressiva

doi: 10.1016/j.omtn.2026.102937

Figure Lengend Snippet: Preferential silencing of ACVR1 R206H in FOP patient-derived fibroblasts using LNA and MOE gapmers (A) Sequence alignment showing LNA and MOE gapmer binding sites spanning the ACVR1 c.617G>A mutation (R206H). Mismatches are shown in lowercase; green: ACVR1 WT variant; red: ACVR1 R206H variant. (B) Schematic overview of the experimental timeline: day 2, cell revival; day 0, seeding; day 1, transfection; day 3, RNA/protein harvest. Toxicity assessments were performed at 4, 12, 24, and 48 h post-transfection. (C and D) RT-qPCR analysis of total ACVR1 mRNA levels following treatment with 100 nM (C) or 10 nM (D) gapmers. (E) Representative immunoblots showing total ACVR1 protein levels post-treatment with 100 and 10 nM gapmers. GAPDH served as a loading control. (F and G) Quantification of total ACVR1 protein abundance relative to healthy control cells at 100 nM (F) and 10 nM (G) using densitometric analysis of the immunoblot images (E). (H–K) Allele-specific RT-qPCR quantification of ACVR1 WT (H, J) and ACVR1 R206H (I, K) transcript levels following treatment with 100 nM (H, I) or 10 nM (J, K) gapmers. (L) Representative immunoblots of V5-tagged ACVR1 WT and ACVR1 R206H proteins expressed in C2C12 cells 48 h post-treatment with 10 nM gapmers, with GAPDH as a loading control. (M and N) Densitometric quantification of immunoblot images (L) showing V5-tagged ACVR1 WT (M) and ACVR1 R206H (N) protein levels in C2C12 cells relative to untreated controls. Statistics, one-way ANOVA with Tukey’s multiple comparisons test; ∗∗∗∗ p < 0.0001. Data are represented as mean ± standard error of the mean ( n = 5–7). NT, non-treated; Lipo, lipofectamine 3000 (a commercially available transfection reagent).

Article Snippet: Murine C2C12 myoblasts (American Type Culture Collection, VA, USA) were cultured in DMEM/F-12 medium supplemented with 15% FBS and 0.5% penicillin-streptomycin under standard conditions.

Techniques: Derivative Assay, Sequencing, Binding Assay, Mutagenesis, Variant Assay, Transfection, Quantitative RT-PCR, Western Blot, Control, Quantitative Proteomics

Journal: Molecular Therapy. Nucleic Acids

Article Title: Single-base 2′OMe-modified LNA and MOE gapmers selectively silence ACVR1 R206H in fibrodysplasia ossificans progressiva

doi: 10.1016/j.omtn.2026.102937

Figure Lengend Snippet: Single-base 2′OMe modification enhances allele selectivity and suppresses ACVR1 R206H -induced osteogenic differentiation in vitro (A and B) RT-qPCR quantification of ACVR1 WT (A) and ACVR1 R206H (B) transcripts in FOP patient-derived fibroblasts treated with 10 nM LNA16 or MOE3 gapmers, with or without a single 2′OMe modification at the second position of the gap region. (C) Allelic fractions of ACVR1 WT and ACVR1 R206H transcripts post-treatment as calculated from total ACVR1 expression. Green and red bars represent ACVR1 WT and ACVR1 R206H transcript fractions, respectively. (D and E) RT-qPCR quantification of ACVR1 WT (D) and ACVR1 R206H (E) transcripts in C2C12 myoblasts transfected with V5-tagged ACVR1 WT or ACVR1 R206H constructs and treated with 10 nM gapmers for 48 h. (F) Allelic fraction analysis of ACVR1 WT and ACVR1 R206H transcripts in cells from (D and E), similarly as (C). (G) Representative immunoblot showing V5-tagged ACVR1 WT and ACVR1 R206H protein levels in transfected C2C12 cells after 10 nM gapmer treatment. GAPDH served as a loading control. (H and I) Densitometric quantification of ACVR1 WT (H) and ACVR1 R206H (I) protein expression from (G), normalized to GAPDH and expressed relative to the non-treated control. (J) Representative images of alkaline phosphatase (ALP) staining in C2C12 cells expressing V5- ACVR1 R206H or ACVR1 WT , treated with 10 nM gapmers and stimulated with recombinant human activin A (100 ng/mL) for 48 h. (K) Quantification of secreted ALP enzymatic activity in conditioned media collected from cells in (J), normalized to the ACVR1 WT media. (L) Representative images of ARS staining performed 21 days after osteogenic differentiation induction in C2C12 cells transfected with V5- ACVR1 R206H or ACVR1 WT and treated with gapmers. (M) Quantification of ARS staining from (L), normalized to the ACVR1 WT control. Statistics, one-way ANOVA with Tukey’s multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Scale bars, 250 μm. Data are represented as mean ± standard error of the mean ( n = 6–7). NT, non-treated; Lipo, lipofectamine 3000 (a commercially available transfection reagent).

Article Snippet: Murine C2C12 myoblasts (American Type Culture Collection, VA, USA) were cultured in DMEM/F-12 medium supplemented with 15% FBS and 0.5% penicillin-streptomycin under standard conditions.

Techniques: Modification, In Vitro, Quantitative RT-PCR, Derivative Assay, Expressing, Transfection, Construct, Western Blot, Control, Staining, Recombinant, Activity Assay