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: bioRxiv

Article Title: Pancreatic cancer extracellular vesicles carry a time-of-day-regulated miRNA cargo that disrupts the skeletal muscle clock and bioenergetics

doi: 10.64898/2026.05.03.722338

Figure Lengend Snippet: (A) Bioluminescence recording, ( B ) period analysis, and ( C ) phase and amplitude analysis of U2OS BMAL1 :Luc reporter cells treated with PANC-1 CM at 12.5%, 25%, 50%, and 100% of the recording media. ( D ) Bioluminescence recording, ( E ) period analysis, and ( F ) phase and amplitude analysis of NIH3T3 Bmal1 :Luc reporter cells treated with PANC-1 CM at the same concentrations. For all bioluminescence experiments, at least three complete oscillations were included in the period estimation, excluding the first 24 h. Mean ± SD of relative mRNA expression of NIH3T3 core clock genes Bmal1 ( G ), Per2 ( H ), and Cry2 ( I ) measured over 36 h in response to PANC-1 CM. Relative mRNA levels of core clock genes in synchronized C2C12 myotubes over 32 h following treatment with PANC-1 CM: ( J ) Bmal1 , ( K ) Per2 , and ( L ) Cry2 . Cosine curves were fit for visualization purposes only; solid lines represent rhythmic oscillations (p<0.05) detected by MetaCycle, while dashed lines indicate loss of statistical rhythmicity (Suppl. Table 1). ( G–L ) Black: Control; ( G–I ) Red: PANC-1 CM; ( J–L ) Blue: PANC-1 CM. ( M ) Schematic representation and representative images of mature C2C12 myotube atrophy in response to NIH3T3 or PANC-1 released factors using a Transwell co-culture system; three measurements per myotube (yellow arrows) were used to quantify shortening. ( N ) Quantification of normalized myotube diameter under NIH3T3 vs PANC-1 co-culture, normalized to NIH3T3 co-culture control. One-way ANOVA: ( B ) p=0.0009, ( E ) p=0.0087. ( B, E ) Dunnett’s post-hoc test: *p<0.05; **p<0.01; ***p<0.001. (N) Student’s t-test: ***p<0.001.

Article Snippet: The human pancreatic cancer cell line PANC-1, the murine fibroblast cell line NIH3T3, and the murine myoblast cell line C2C12 were purchased from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Control, Co-Culture Assay

Journal: bioRxiv

Article Title: Pancreatic cancer extracellular vesicles carry a time-of-day-regulated miRNA cargo that disrupts the skeletal muscle clock and bioenergetics

doi: 10.64898/2026.05.03.722338

Figure Lengend Snippet: (A) Top 35 miRNAs by mean expression in PANC-1-derived sEVs. Bar plot of mean log2 CPM across 9 time-points (4–36 h). Red bars: miRNAs selected from the top 35 to be tested in the BMAL1 :Luc reporter and atrophy assays; grey bars: remaining top-35 miRNAs. miRNAs are ranked in descending order of EV expression. ( B ) GO Biological Process enrichment of the experimentally validated targets (miRTarBase) of the 11 selected miRNAs. Terms are grouped into functional categories. Dot size represents the number of validated target genes associated with each term; dot color indicates Gene Ratio (proportion of input genes annotated to the term), from light pink (low) to dark red (high). Analysis performed with clusterProfiler. ( C , top panel) Normalized C2C12 myotube diameter at 0, 24, and 48 h post-transfection with miR-27b-3p, miR-615-3p, miR-191-5p, miR-127-3p, miR-99b-5p, or negative-transfection control (NTC); dexamethasone (Dexa) included as positive control. ( C , lower panel) Normalized C2C12 myotube diameter at the same time-points after transfection with hsa-let-7f-5p, miR-183-5p, miR-92a-3p, miR-30c-5p, miR-26a-5p, miR-10a-5p, NTC, or Dexa. ( D ) Oxygen consumption rate (OCR; top), resting-phenotype plot of basal OCR vs ECAR (middle), and metabolic-capacity plot of maximal OCR vs ECAR following FCCP (lower) for mature C2C12 myotubes 48 h post-transfection with miR-27b-3p, miR-615-3p, miR-191-5p, or NTC (Control). ( E ) Same panels for myotubes transfected with miR-127-3p, miR-99b-5p, miR-183-5p, or NTC. Sequential injections of oligomycin, FCCP, and rotenone/antimycin A were used to dissect mitochondrial respiration. Data are presented as mean ± SEM. ( C ) Measurements were taken from at least 5 random fields per well in N=3 wells; statistical analysis used 2-way ANOVA with Dunnett’s post-hoc correction: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: The human pancreatic cancer cell line PANC-1, the murine fibroblast cell line NIH3T3, and the murine myoblast cell line C2C12 were purchased from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Derivative Assay, Functional Assay, Transfection, Control, Positive Control