Journal: Advanced Science

Article Title: Ultrafast Micrometric Laser Structuring of Polymer Surfaces for Highly Organized Skeletal Muscle Tissue Modeling

doi: 10.1002/advs.78129

Figure Lengend Snippet: Relative gene expression of C2C12 myoblasts cultured on the LS surface compared with the standard surface. Data are presented as mean ± SD with individual data points ( n = 3). Gene expression levels were normalized to the housekeeping gene and expressed as fold change relative to the standard group (Std). Panels correspond to: (a) d0, (b) d4, (c) d7, and (d) d11. Statistical significance was determined using Student's t ‐test: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. Standard.

Article Snippet: Murine myoblasts C2C12 (ATCC) were cultured according to a previously published protocol [ ] in T‐75 Flasks (Sarstedt) at 37°C and 5% CO 2 in Dulbecco's modified Eagle medium GlutaMAX (DMEM, Gibco) supplemented with 10% heat‐inactivated fetal bovine serum (FBS, EuroClone), penicillin (100 IU mL −1 , Gibco), and streptomycin (100 mg mL −1 , Gibco).

Techniques: Gene Expression, Cell Culture

Journal: Advanced Science

Article Title: Ultrafast Micrometric Laser Structuring of Polymer Surfaces for Highly Organized Skeletal Muscle Tissue Modeling

doi: 10.1002/advs.78129

Figure Lengend Snippet: (a) Infrared spectra of the standard surface before (Std, red line) and after laser structuring (LS, blue line); (b) MTT assay. The C2C12 cell growth at three experimental time points (24 h, 48 h, and 72 h) is plotted for the cells seeded on a standard surface (Std, red line) and LS (ex. LS, blue line) surfaces. A theoretical curve was also plotted, taking into account the area available on the LS surface (th. LS, dashed dark blue line). (a.u. = arbitrary unit); (c) Brightfield pictures of the cells cultured on standard (top pictures) or LS (bottom pictures) surfaces at the three experimental time points (24 h, 48 h, and 72 h).

Article Snippet: Murine myoblasts C2C12 (ATCC) were cultured according to a previously published protocol [ ] in T‐75 Flasks (Sarstedt) at 37°C and 5% CO 2 in Dulbecco's modified Eagle medium GlutaMAX (DMEM, Gibco) supplemented with 10% heat‐inactivated fetal bovine serum (FBS, EuroClone), penicillin (100 IU mL −1 , Gibco), and streptomycin (100 mg mL −1 , Gibco).

Techniques: Infrared, MTT Assay, Brightfield, Cell Culture

Journal: Advanced Science

Article Title: Ultrafast Micrometric Laser Structuring of Polymer Surfaces for Highly Organized Skeletal Muscle Tissue Modeling

doi: 10.1002/advs.78129

Figure Lengend Snippet: Image analyses conducted on C2C12 after 7 days of differentiation. Pictures a, b, and c show representative immunofluorescence pictures superimposed on the top with their relative brightfield pictures. The myotubes positive for MHC (Red), are randomly oriented in the standard plates (a) while properly aligned on the LS surfaces (channel spacing of 75 µm (b) and 150 µm (c)). Orientation of muscle fibers. The distribution of orientation is plotted for unstructured standard plates (Std) and for all spacing (d, 75, 100, 125, and 150 µm) and depth (g, 3.5 – 5 – 8;5 – 11.5 – 14 – 15 ‐20 µm) values tested, revealing the presence of a prevalent peak for all conditions compared to their respective control (black plot); the quantification of the orientation through the coherency value (e, f) shows a significative increase for all the LS surfaces compared to the standard ( * ). Moreover, regarding the spacing analysis, the three higher values are also significative compared to 75 µm (e, $ ). Myotube lengths. The myotubes differentiated on LS surfaces with a spacing of 125 µm and a depth of 11.5 µm (h, red bar) are significantly longer than those grown on the standard plates (h, black bar). Sample size n = 3. p value list: spacing, **** p < 0.0001, *** p = 0.0001, $$$ p = 0.0006, $$ p = 0.0013 (75 µm vs 100 µm), $$ p = 0.0059 (75 µm vs 150 µm); depth, **** p < 0.0001, *** p = 0.0007, ** p = 0.001 (3.5 µm), ** p = 0.0025 (14 µm), ** p = 0.0085 (20 µm); length, **** p < 0.0001.

Article Snippet: Murine myoblasts C2C12 (ATCC) were cultured according to a previously published protocol [ ] in T‐75 Flasks (Sarstedt) at 37°C and 5% CO 2 in Dulbecco's modified Eagle medium GlutaMAX (DMEM, Gibco) supplemented with 10% heat‐inactivated fetal bovine serum (FBS, EuroClone), penicillin (100 IU mL −1 , Gibco), and streptomycin (100 mg mL −1 , Gibco).

Techniques: Immunofluorescence, Brightfield, Control, Analysis

Journal: Advanced Science

Article Title: Ultrafast Micrometric Laser Structuring of Polymer Surfaces for Highly Organized Skeletal Muscle Tissue Modeling

doi: 10.1002/advs.78129

Figure Lengend Snippet: Analysis of MHC and Desmin protein levels during C2C12 myoblast differentiation. Western blot analysis of C2C12 cells cultured on standard (Std) and LS surfaces at different stages of differentiation. Representative immunoblot for (a) MHC and Tubulin, and (c) Desmin and GAPDH. Densitometric analysis of (b) MHC and (d) Desmin protein levels. Data are presented as mean ± SD with individual data points ( n = 3). Statistical significance was assessed using Student's t ‐test ( * p < 0.05; ** p < 0.01; *** p < 0.001 vs. Standard at each time point).

Article Snippet: Murine myoblasts C2C12 (ATCC) were cultured according to a previously published protocol [ ] in T‐75 Flasks (Sarstedt) at 37°C and 5% CO 2 in Dulbecco's modified Eagle medium GlutaMAX (DMEM, Gibco) supplemented with 10% heat‐inactivated fetal bovine serum (FBS, EuroClone), penicillin (100 IU mL −1 , Gibco), and streptomycin (100 mg mL −1 , Gibco).

Techniques: Analysis, Western Blot, Cell Culture

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