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DFO treatment had no toxic or off target effects on C2C12 <t>myotubes.</t> (A) Quantification of total nuclei within myotubes (ANOVA; F (3, 8) = 7.248, p = 0.0114, η 2 = 0.731) (N = 3) (B) Representative analysis images in each group generated by the MyoCount program, used to quantify total nuclei within myotubes. Green, myotubes. Blue dots, nuclei. (C) Representative images of C2C12 myotubes after treatment with DFO, DFO + FeCl 3 , or control. Scale bars, 500 μm. Magnification, 100x. (D) Quantification of OD583nm/1.5 × 10 6 cells (ANOVA; F (2, 6) = 3166, p < 0.001, η 2 = 0.999) (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments, and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. NFI, nuclear fusion index.

Myotubes, supplied by Tokyo Chemical Industry, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy"

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2026.102451

DFO treatment had no toxic or off target effects on C2C12 myotubes. (A) Quantification of total nuclei within myotubes (ANOVA; F (3, 8) = 7.248, p = 0.0114, η 2 = 0.731) (N = 3) (B) Representative analysis images in each group generated by the MyoCount program, used to quantify total nuclei within myotubes. Green, myotubes. Blue dots, nuclei. (C) Representative images of C2C12 myotubes after treatment with DFO, DFO + FeCl 3 , or control. Scale bars, 500 μm. Magnification, 100x. (D) Quantification of OD583nm/1.5 × 10 6 cells (ANOVA; F (2, 6) = 3166, p < 0.001, η 2 = 0.999) (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments, and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. NFI, nuclear fusion index.

Figure Legend Snippet: DFO treatment had no toxic or off target effects on C2C12 myotubes. (A) Quantification of total nuclei within myotubes (ANOVA; F (3, 8) = 7.248, p = 0.0114, η 2 = 0.731) (N = 3) (B) Representative analysis images in each group generated by the MyoCount program, used to quantify total nuclei within myotubes. Green, myotubes. Blue dots, nuclei. (C) Representative images of C2C12 myotubes after treatment with DFO, DFO + FeCl 3 , or control. Scale bars, 500 μm. Magnification, 100x. (D) Quantification of OD583nm/1.5 × 10 6 cells (ANOVA; F (2, 6) = 3166, p < 0.001, η 2 = 0.999) (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments, and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. NFI, nuclear fusion index.

Techniques Used: Generated, Control

BCAA had minimal effects on myotube diameter. (A) Representative immunocytochemical images of myotubes. Green: Myotubes (MyHC staining). Blue: Nuclei (DAPI staining). Scale bars, 100 μm. Magnification, 200x. (B) Quantification of the myotube diameter (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments, and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes (ANOVA; F (3, 8) = 8.454, p = 0.007, η 2 = 0.760), followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. (C) The violin plot of the relationships between myotube diameter and four experimental groups. The distribution of data in each group was evaluated. The horizontal axis presents the myotube diameter while the vertical axis presents four experimental groups. (D) Quantification of NFI (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes (ANOVA; F (3, 8) = 9.374, p = 0.005, η 2 = 0.779), followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. NFI, nuclear fusion index.

Figure Legend Snippet: BCAA had minimal effects on myotube diameter. (A) Representative immunocytochemical images of myotubes. Green: Myotubes (MyHC staining). Blue: Nuclei (DAPI staining). Scale bars, 100 μm. Magnification, 200x. (B) Quantification of the myotube diameter (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments, and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes (ANOVA; F (3, 8) = 8.454, p = 0.007, η 2 = 0.760), followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. (C) The violin plot of the relationships between myotube diameter and four experimental groups. The distribution of data in each group was evaluated. The horizontal axis presents the myotube diameter while the vertical axis presents four experimental groups. (D) Quantification of NFI (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes (ANOVA; F (3, 8) = 9.374, p = 0.005, η 2 = 0.779), followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. NFI, nuclear fusion index.

Techniques Used: Staining

BCAA reduced Atrogin-1 expression in DFO-treated myotubes, but had no effect on MuRF-1 expression. (A) Relative expression of Atrogin-1(F (3, 8) = 46.837, p < 0.001, η 2 = 0.946) (N = 3). (B) Relative expression of MuRF-1(F (3, 8) = 26.29, p < 0.001, η 2 = 0.908) (N = 3). (C) (D) Representative WB blot images showing the protein expression of Atrogin-1 and MuRF-1. Protein was extracted on 24 h after BCAA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments, and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure Legend Snippet: BCAA reduced Atrogin-1 expression in DFO-treated myotubes, but had no effect on MuRF-1 expression. (A) Relative expression of Atrogin-1(F (3, 8) = 46.837, p < 0.001, η 2 = 0.946) (N = 3). (B) Relative expression of MuRF-1(F (3, 8) = 26.29, p < 0.001, η 2 = 0.908) (N = 3). (C) (D) Representative WB blot images showing the protein expression of Atrogin-1 and MuRF-1. Protein was extracted on 24 h after BCAA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments, and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Techniques Used: Expressing

BCAA increased the p-Akt in DFO-treated myotubes after 45 min of BCAA treatment, but had no statistically significant effects on other signaling molecules at the same time point. Representative blots for quantification of (A) p-Akt (F (3, 8) = 6.166, p = 0.018, η 2 = 0.6981), p-mTOR, p-p70S6K, p-4E-BP1, and p-eEF2, (B) p-AMPK, and p-ACC, and (C) p-FOXO1, and p–NF–κB p65. Protein was extracted at 45 min after BCCA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments (N = 3), and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05.

Figure Legend Snippet: BCAA increased the p-Akt in DFO-treated myotubes after 45 min of BCAA treatment, but had no statistically significant effects on other signaling molecules at the same time point. Representative blots for quantification of (A) p-Akt (F (3, 8) = 6.166, p = 0.018, η 2 = 0.6981), p-mTOR, p-p70S6K, p-4E-BP1, and p-eEF2, (B) p-AMPK, and p-ACC, and (C) p-FOXO1, and p–NF–κB p65. Protein was extracted at 45 min after BCCA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments (N = 3), and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05.

Techniques Used:

BCAA increased the phosphorylation of p70S6K in DFO-treated myotubes after 24 h of BCAA treatment, but had no effect on the phosphorylation of AMPK, Akt, or eEF2. (A) Representative blots for quantification of (B) p-AMPK(F (3, 8) = 5.620, p = 0.023, η 2 = 0.678), (C) p-Akt (F (3, 8) = 6.394, p = 0.016, η 2 = 0.706), (D) p-p70S6K(F (3, 8) = 16.43, p < 0.001, η 2 = 0.860), and (E) p-eEF2 (F (3, 8) = 10.23, p = 0.004, η 2 = 0.793). Protein was extracted 24 h after BCCA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments (N = 3), and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗p < 0.01.

Figure Legend Snippet: BCAA increased the phosphorylation of p70S6K in DFO-treated myotubes after 24 h of BCAA treatment, but had no effect on the phosphorylation of AMPK, Akt, or eEF2. (A) Representative blots for quantification of (B) p-AMPK(F (3, 8) = 5.620, p = 0.023, η 2 = 0.678), (C) p-Akt (F (3, 8) = 6.394, p = 0.016, η 2 = 0.706), (D) p-p70S6K(F (3, 8) = 16.43, p < 0.001, η 2 = 0.860), and (E) p-eEF2 (F (3, 8) = 10.23, p = 0.004, η 2 = 0.793). Protein was extracted 24 h after BCCA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments (N = 3), and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗p < 0.01.

Techniques Used: Phospho-proteomics

Concentration Assay:

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy
Article Snippet: After five days (designated as Day 0) of culture in DM, the iron chelator deferoxamine (DFO; #D9533, Sigma-Aldrich), which was applied by previous studies to establish an iron deficient in vitro model, was administered at a concentration of 100 μM in the DFO and DFO + BCAA groups for 2 or 4 days [ , ]. .. For BCAA stimulation, serum and BCAA were excluded from the medium for 90 min to starve myotubes, then BCAA ( l -Leucine, l -Isoleucine, and l -Valine, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added at a concentration of 5 mM ( l -leucine: l -isoleucine: l -valine = 2:1:1) for 45 min or 24 h before harvesting cells for protein extraction [ , ]. ..

Protein Extraction:

Image Search Results

(A) RT-PCR analysis of GIPR and GLP-1R expression in differentiated primary human myotubes from six osteoarthritis patients (three technical replicates per donor). GIPR was consistently detected across all donors; GLP-1R was undetectable in all samples. (B) CT and ΔCT values for GIPR and GLP-1R across all six donors (ΔCT values: 35.00 ± 0.81, 33.07 ± 0.71, 35.01 ± 0.73, 34.85 ± 1.21, 35.80 ± 0.54, 33.36 ± 0.23; mean ± SD). (C) Mean fluorescence intensity (MFI) fold change relative to unstained controls in human primary myoblasts live-stained with LUXendin645 (500 nM, 15 minutes). Data from three donors shown with individual patient values connected by lines; bar represents the group mean. *p = 0.0383, paired t-test. (D) Representative gating strategy for LUXendin645 flow cytometry on the BD Accuri C6 Plus. Upper row: unstained human primary myoblasts. Lower row: LUXendin645-stained human primary myoblasts. Sequential gating: FSC/SSC scatter to exclude debris, (2) singlet discrimination, (3 & 4) fluorescent signal as histogram and dot plot to identify LUXendin645-positive cells. (E) MFI values and percentage LUXendin645-positive cells for each of the three donors.

Journal: bioRxiv

Article Title: GLP-1 and GIP receptor agonism does not directly drive skeletal muscle atrophy or impair myogenesis in primary human myotubes

doi: 10.64898/2026.07.20.739515

Figure Lengend Snippet: (A) RT-PCR analysis of GIPR and GLP-1R expression in differentiated primary human myotubes from six osteoarthritis patients (three technical replicates per donor). GIPR was consistently detected across all donors; GLP-1R was undetectable in all samples. (B) CT and ΔCT values for GIPR and GLP-1R across all six donors (ΔCT values: 35.00 ± 0.81, 33.07 ± 0.71, 35.01 ± 0.73, 34.85 ± 1.21, 35.80 ± 0.54, 33.36 ± 0.23; mean ± SD). (C) Mean fluorescence intensity (MFI) fold change relative to unstained controls in human primary myoblasts live-stained with LUXendin645 (500 nM, 15 minutes). Data from three donors shown with individual patient values connected by lines; bar represents the group mean. *p = 0.0383, paired t-test. (D) Representative gating strategy for LUXendin645 flow cytometry on the BD Accuri C6 Plus. Upper row: unstained human primary myoblasts. Lower row: LUXendin645-stained human primary myoblasts. Sequential gating: FSC/SSC scatter to exclude debris, (2) singlet discrimination, (3 & 4) fluorescent signal as histogram and dot plot to identify LUXendin645-positive cells. (E) MFI values and percentage LUXendin645-positive cells for each of the three donors.

Article Snippet: Differentiated human skeletal muscle myotubes (n = 6 donors) were treated with semaglutide (10 nM; MedChemExpress HY-114118; DMSO vehicle) or GIP peptide (5.02 μM; MedChemExpress HY-P0276; H 2 O vehicle).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence, Staining, Flow Cytometry

Data from n = 6 donors unless otherwise stated. All PCR comparisons by Wilcoxon signed-rank test; ns = not significant. (A) mRNA expression (fold change to vehicle control) of atrophy- and inflammation-related genes in human primary myotubes 4 hours after treatment with semaglutide vs DMSO (left) or GIP vs H ₂ O (right). Genes assessed: GIPR, MAFBX, MURF1, FOXO3, IL6, and TGFB. GLP-1R was also assessed but was undetectable in all samples, consistent with . (B) Principal component analysis (PCA) of Olink Reveal normalised protein expression (NPX) data from primary human myotubes treated with GIP (H ₂ O vehicle control), semaglutide (DMSO vehicle control), or their respective vehicle controls. Points represent individual samples; 95% confidence ellipses are shown for each treatment group. (C) Volcano plots showing differential protein expression between GIP vs H ₂ O vehicle control (left) and semaglutide vs DMSO vehicle control (right). The x-axis shows log ₂ fold change (NPX) and the y-axis shows −log ₁₀ (nominal p-value) from Welch’s two-sample t- test. Dashed lines indicate thresholds of |log ₂ FC| > 0.58 and p < 0.05 (unadjusted). No proteins reached statistical significance after Benjamini–Hochberg correction across 1,034 proteins tested (minimum BH-adjusted p = 1.0, both comparisons). At nominal thresholds, three proteins were upregulated in GIP-treated myotubes (UBE2B, S100A12, CRNN), and one following semaglutide treatment (CD79B). (D) Heatmaps showing z-scored NPX values for the 100 proteins with the lowest nominal p- values from GIP vs H ₂ O comparison (left) and semaglutide vs DMSO comparison (right). Rows are hierarchically clustered; columns ordered by treatment group. Colour scale: red = high NPX, blue = low NPX. (E) Individual delta NPX values (NPX − mean NPX of vehicle control) for six proteins of functional relevance to muscle biology: FOXO1, MSTN, IGF1R, TGFB1, FOXO3, and IL6. Data shown for semaglutide vs DMSO vehicle control and GIP vs H ₂ O vehicle control. Individual donor values are plotted with mean ± SEM.

Journal: bioRxiv

Article Title: GLP-1 and GIP receptor agonism does not directly drive skeletal muscle atrophy or impair myogenesis in primary human myotubes

doi: 10.64898/2026.07.20.739515

Figure Lengend Snippet: Data from n = 6 donors unless otherwise stated. All PCR comparisons by Wilcoxon signed-rank test; ns = not significant. (A) mRNA expression (fold change to vehicle control) of atrophy- and inflammation-related genes in human primary myotubes 4 hours after treatment with semaglutide vs DMSO (left) or GIP vs H ₂ O (right). Genes assessed: GIPR, MAFBX, MURF1, FOXO3, IL6, and TGFB. GLP-1R was also assessed but was undetectable in all samples, consistent with . (B) Principal component analysis (PCA) of Olink Reveal normalised protein expression (NPX) data from primary human myotubes treated with GIP (H ₂ O vehicle control), semaglutide (DMSO vehicle control), or their respective vehicle controls. Points represent individual samples; 95% confidence ellipses are shown for each treatment group. (C) Volcano plots showing differential protein expression between GIP vs H ₂ O vehicle control (left) and semaglutide vs DMSO vehicle control (right). The x-axis shows log ₂ fold change (NPX) and the y-axis shows −log ₁₀ (nominal p-value) from Welch’s two-sample t- test. Dashed lines indicate thresholds of |log ₂ FC| > 0.58 and p < 0.05 (unadjusted). No proteins reached statistical significance after Benjamini–Hochberg correction across 1,034 proteins tested (minimum BH-adjusted p = 1.0, both comparisons). At nominal thresholds, three proteins were upregulated in GIP-treated myotubes (UBE2B, S100A12, CRNN), and one following semaglutide treatment (CD79B). (D) Heatmaps showing z-scored NPX values for the 100 proteins with the lowest nominal p- values from GIP vs H ₂ O comparison (left) and semaglutide vs DMSO comparison (right). Rows are hierarchically clustered; columns ordered by treatment group. Colour scale: red = high NPX, blue = low NPX. (E) Individual delta NPX values (NPX − mean NPX of vehicle control) for six proteins of functional relevance to muscle biology: FOXO1, MSTN, IGF1R, TGFB1, FOXO3, and IL6. Data shown for semaglutide vs DMSO vehicle control and GIP vs H ₂ O vehicle control. Individual donor values are plotted with mean ± SEM.

Techniques: Expressing, Control, Comparison, Functional Assay

(A) Representative desmin/DAPI immunofluorescence images of human primary myoblasts before and after differentiation. Undifferentiated myoblasts appear as long, thin, mononucleated cells; differentiated myotubes are thicker and multinucleated. (B) Representative desmin/DAPI images of differentiated myotubes treated with or without TNFα for 24 hours. TNFα treatment induced visible myotube thinning while preserving multinucleation, confirming TNFα as a positive control for atrophy induction. (C) Representative desmin/DAPI images of control myotubes and myotubes following 24 hours of incretin treatment. (D) Myotube thickness (µm) following 24 hours of treatment with semaglutide vs vehicle (DMSO; left) or GIP vs vehicle (H ₂ O; right).

Journal: bioRxiv

Article Title: GLP-1 and GIP receptor agonism does not directly drive skeletal muscle atrophy or impair myogenesis in primary human myotubes

doi: 10.64898/2026.07.20.739515

Figure Lengend Snippet: (A) Representative desmin/DAPI immunofluorescence images of human primary myoblasts before and after differentiation. Undifferentiated myoblasts appear as long, thin, mononucleated cells; differentiated myotubes are thicker and multinucleated. (B) Representative desmin/DAPI images of differentiated myotubes treated with or without TNFα for 24 hours. TNFα treatment induced visible myotube thinning while preserving multinucleation, confirming TNFα as a positive control for atrophy induction. (C) Representative desmin/DAPI images of control myotubes and myotubes following 24 hours of incretin treatment. (D) Myotube thickness (µm) following 24 hours of treatment with semaglutide vs vehicle (DMSO; left) or GIP vs vehicle (H ₂ O; right).

Techniques: Immunofluorescence, Preserving, Positive Control, Control

Data shown are from n = 3 biological replicates (PCR) or n = 6 biological replicates (all other assays). Myotubes were treated with semaglutide or GIP (with appropriate vehicle controls) every 2 days during the 8-day differentiation protocol. All comparisons by Wilcoxon signed-rank test; ns = not significant. (A) MyoG, MyoD, and Myf5 mRNA expression (fold change to vehicle control) at Day 2 and Day 4 of differentiation, for semaglutide vs vehicle control (left, blue) and GIP vs vehicle control (right, red). (B) Nuclear fusion index (NFI, %) following 8 days of differentiation with semaglutide vs DMSO (left) and GIP vs H ₂ O (right). Representative desmin/DAPI immunofluorescence images are shown for each condition (DMSO, semaglutide, H ₂ O, GIP). (C) Myotube coverage (%) following 8 days of differentiation. (D) Representative images used for lactate dehydrogenase (LDH) quantification, following 8 days of differentiation.| (E) LDH release (absorbance 450–650 nm) following 8 days of differentiation, shown for all four conditions. LDH release was consistent between semaglutide, DMSO, and GIP, but significantly elevated in the H ₂ O vehicle control condition relative to all three (vs DMSO, p = 0.0014; vs semaglutide, p = 0.0004; vs GIP, p = 0.0003), consistent with a hypotonic vehicle effect rather than a treatment effect. (F) Myotube thickness (µm) following 8 days of differentiation with semaglutide vs DMSO (left) and GIP vs H ₂ O (right). Semaglutide p = 0.7708; GIP p = 0.0205.

Journal: bioRxiv

Article Title: GLP-1 and GIP receptor agonism does not directly drive skeletal muscle atrophy or impair myogenesis in primary human myotubes

doi: 10.64898/2026.07.20.739515

Figure Lengend Snippet: Data shown are from n = 3 biological replicates (PCR) or n = 6 biological replicates (all other assays). Myotubes were treated with semaglutide or GIP (with appropriate vehicle controls) every 2 days during the 8-day differentiation protocol. All comparisons by Wilcoxon signed-rank test; ns = not significant. (A) MyoG, MyoD, and Myf5 mRNA expression (fold change to vehicle control) at Day 2 and Day 4 of differentiation, for semaglutide vs vehicle control (left, blue) and GIP vs vehicle control (right, red). (B) Nuclear fusion index (NFI, %) following 8 days of differentiation with semaglutide vs DMSO (left) and GIP vs H ₂ O (right). Representative desmin/DAPI immunofluorescence images are shown for each condition (DMSO, semaglutide, H ₂ O, GIP). (C) Myotube coverage (%) following 8 days of differentiation. (D) Representative images used for lactate dehydrogenase (LDH) quantification, following 8 days of differentiation.| (E) LDH release (absorbance 450–650 nm) following 8 days of differentiation, shown for all four conditions. LDH release was consistent between semaglutide, DMSO, and GIP, but significantly elevated in the H ₂ O vehicle control condition relative to all three (vs DMSO, p = 0.0014; vs semaglutide, p = 0.0004; vs GIP, p = 0.0003), consistent with a hypotonic vehicle effect rather than a treatment effect. (F) Myotube thickness (µm) following 8 days of differentiation with semaglutide vs DMSO (left) and GIP vs H ₂ O (right). Semaglutide p = 0.7708; GIP p = 0.0205.

Techniques: Expressing, Control, Immunofluorescence

FAPs accounted for the disparity in NMJ regeneration between MAS and TA muscles. (a) AChR and MyHC immunolabelling in myotubes differentiated from MuSCs isolated from MAS or TA muscles. Mature myotubes were treated with or without agrin (100 ng/mL) for 16 h. Scale bar = 50 μm. Graphs in the right panel show the quantifications of differentiation index (upper left), myotube width (upper right), AChR cluster counts normalized to myotube area (lower left), ratio of AChR/myotube area (lower middle) and Chrna1 expression quantified by real‐time quantitative PCR (lower right). n = 3. (b) AChR and MyHC staining of MuSCs single culture and cocultures with different FAPs. Scale bar = 50 μm. Graphs in the lower panel show (from left to right) the quantification of differentiation index, myotube area, AChR cluster density and ratio of AChR/myotube area. n = 3. (c) Schematic of the direct coculture of different FAPs with MAS‐derived MuSCs. (d) Representative images of tdTomato + FAPs and α‐BTX labelled AChRs (green) in recipient MAS and TA muscle. Scale bar = 20 μm. (e) Schematic of the FAPs transplantation assay. (f) Quantification of BTX signals per field. n = 6 mice/group. The data are shown as mean ± SD. Unpaired Student's t test was used in (a, upper right panel). Two‐way ANOVA followed by Tukey's post hoc test was used in (a, lower right panel and f). One‐way ANOVA followed by Tukey's post hoc test was used in (b). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: FAPs accounted for the disparity in NMJ regeneration between MAS and TA muscles. (a) AChR and MyHC immunolabelling in myotubes differentiated from MuSCs isolated from MAS or TA muscles. Mature myotubes were treated with or without agrin (100 ng/mL) for 16 h. Scale bar = 50 μm. Graphs in the right panel show the quantifications of differentiation index (upper left), myotube width (upper right), AChR cluster counts normalized to myotube area (lower left), ratio of AChR/myotube area (lower middle) and Chrna1 expression quantified by real‐time quantitative PCR (lower right). n = 3. (b) AChR and MyHC staining of MuSCs single culture and cocultures with different FAPs. Scale bar = 50 μm. Graphs in the lower panel show (from left to right) the quantification of differentiation index, myotube area, AChR cluster density and ratio of AChR/myotube area. n = 3. (c) Schematic of the direct coculture of different FAPs with MAS‐derived MuSCs. (d) Representative images of tdTomato + FAPs and α‐BTX labelled AChRs (green) in recipient MAS and TA muscle. Scale bar = 20 μm. (e) Schematic of the FAPs transplantation assay. (f) Quantification of BTX signals per field. n = 6 mice/group. The data are shown as mean ± SD. Unpaired Student's t test was used in (a, upper right panel). Two‐way ANOVA followed by Tukey's post hoc test was used in (a, lower right panel and f). One‐way ANOVA followed by Tukey's post hoc test was used in (b). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Muscles, Isolation, Expressing, Real-time Polymerase Chain Reaction, Staining, Derivative Assay, Transplantation Assay

MSTN was the paracrine factor that distinguishes MAS‐derived FAPs from TA‐derived FAPs. (a) AChR and MyHC staining of MuSC single culture or cocultures with conditioned medium (CM) of MAS‐ or TA‐isolated FAPs. Scale bar = 50 μm. (b) Graphs show (from left to right) the quantification of myotube area, average AChR cluster size, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 6. (c) Venn diagram depicting the overlap among DEGs between 7 dpi MAS‐ and TA‐derived FAPs (orange), NMJ‐regulating genes (green) and genes encoding secreted proteins (blue). (d) Mstn expression levels were quantified by qPCR. n = 3. (e) Myostatin levels were quantified by ELISA in CM from 7 dpi MAS‐ or TA‐derived FAPs. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used in (b). Unpaired Student's t test was used in (d,e). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: MSTN was the paracrine factor that distinguishes MAS‐derived FAPs from TA‐derived FAPs. (a) AChR and MyHC staining of MuSC single culture or cocultures with conditioned medium (CM) of MAS‐ or TA‐isolated FAPs. Scale bar = 50 μm. (b) Graphs show (from left to right) the quantification of myotube area, average AChR cluster size, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 6. (c) Venn diagram depicting the overlap among DEGs between 7 dpi MAS‐ and TA‐derived FAPs (orange), NMJ‐regulating genes (green) and genes encoding secreted proteins (blue). (d) Mstn expression levels were quantified by qPCR. n = 3. (e) Myostatin levels were quantified by ELISA in CM from 7 dpi MAS‐ or TA‐derived FAPs. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used in (b). Unpaired Student's t test was used in (d,e). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Derivative Assay, Staining, Isolation, Expressing, Enzyme-linked Immunosorbent Assay

Recombinant MSTN suppressed AChR clustering and miR‐206 expression. (a) AChR and MyHC staining of MuSCs cultures treated with or without recombinant MSTN (100 ng/mL) and follistatin (500 ng/mL). Scale bar = 50μm. (b) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. (c) The levels of miR‐206 expression in MuSCs cultures. Mature myotubes differentiated from MAS‐derived MuSCs were treated with control, agrin alone, agrin plus MSTN or agrin plus MSTN and follistatin. (d) AChR and MyHC staining of myotubes transfected with miR‐206 mimics or negative control (NC) prior to treatment with agrin and MSTN. Scale bar = 50 μm. (e) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: Recombinant MSTN suppressed AChR clustering and miR‐206 expression. (a) AChR and MyHC staining of MuSCs cultures treated with or without recombinant MSTN (100 ng/mL) and follistatin (500 ng/mL). Scale bar = 50μm. (b) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. (c) The levels of miR‐206 expression in MuSCs cultures. Mature myotubes differentiated from MAS‐derived MuSCs were treated with control, agrin alone, agrin plus MSTN or agrin plus MSTN and follistatin. (d) AChR and MyHC staining of myotubes transfected with miR‐206 mimics or negative control (NC) prior to treatment with agrin and MSTN. Scale bar = 50 μm. (e) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Recombinant, Expressing, Staining, Derivative Assay, Control, Transfection, Negative Control

MSTN knockdown reversed the negative effect of MAS FAPs on in vitro AChR clustering. (a) MSTN protein and mRNA level in 7 dpi MAS FAPs transfected with negative control (pGPU6/GFP/Neo‐NC) or knockdown vectors (pGPU6/GFP/Neo‐shMSTN). (b) MSTN protein and mRNA level in 7 dpi TA FAPs transfected with negative control (pcDNA3.1(+)) or overexpression vectors (pcDNA3.1(+)‐MSTN). (c) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster counts normalized to myotube area (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN KD FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (d) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster density (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN OE FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (e,f) Transcript levels of Chrna1 , Chrne , Chrng , Musk and Rapsn in myotubes treated with CM from NC or KD FAPs, and NC or OE FAPs. KD, knock down; OE, overexpression. The data are shown as mean ± SD. Unpaired Student's t test was used in (a–d). Two‐way ANOVA followed by Sidak post hoc test was used in (e,f). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: MSTN knockdown reversed the negative effect of MAS FAPs on in vitro AChR clustering. (a) MSTN protein and mRNA level in 7 dpi MAS FAPs transfected with negative control (pGPU6/GFP/Neo‐NC) or knockdown vectors (pGPU6/GFP/Neo‐shMSTN). (b) MSTN protein and mRNA level in 7 dpi TA FAPs transfected with negative control (pcDNA3.1(+)) or overexpression vectors (pcDNA3.1(+)‐MSTN). (c) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster counts normalized to myotube area (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN KD FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (d) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster density (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN OE FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (e,f) Transcript levels of Chrna1 , Chrne , Chrng , Musk and Rapsn in myotubes treated with CM from NC or KD FAPs, and NC or OE FAPs. KD, knock down; OE, overexpression. The data are shown as mean ± SD. Unpaired Student's t test was used in (a–d). Two‐way ANOVA followed by Sidak post hoc test was used in (e,f). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Knockdown, In Vitro, Transfection, Negative Control, Over Expression, Staining, Derivative Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: DFO treatment had no toxic or off target effects on C2C12 myotubes. (A) Quantification of total nuclei within myotubes (ANOVA; F (3, 8) = 7.248, p = 0.0114, η 2 = 0.731) (N = 3) (B) Representative analysis images in each group generated by the MyoCount program, used to quantify total nuclei within myotubes. Green, myotubes. Blue dots, nuclei. (C) Representative images of C2C12 myotubes after treatment with DFO, DFO + FeCl 3 , or control. Scale bars, 500 μm. Magnification, 100x. (D) Quantification of OD583nm/1.5 × 10 6 cells (ANOVA; F (2, 6) = 3166, p < 0.001, η 2 = 0.999) (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments, and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. NFI, nuclear fusion index.

Article Snippet: For BCAA stimulation, serum and BCAA were excluded from the medium for 90 min to starve myotubes, then BCAA ( l -Leucine, l -Isoleucine, and l -Valine, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added at a concentration of 5 mM ( l -leucine: l -isoleucine: l -valine = 2:1:1) for 45 min or 24 h before harvesting cells for protein extraction [ , ].

Techniques: Generated, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: BCAA had minimal effects on myotube diameter. (A) Representative immunocytochemical images of myotubes. Green: Myotubes (MyHC staining). Blue: Nuclei (DAPI staining). Scale bars, 100 μm. Magnification, 200x. (B) Quantification of the myotube diameter (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments, and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes (ANOVA; F (3, 8) = 8.454, p = 0.007, η 2 = 0.760), followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. (C) The violin plot of the relationships between myotube diameter and four experimental groups. The distribution of data in each group was evaluated. The horizontal axis presents the myotube diameter while the vertical axis presents four experimental groups. (D) Quantification of NFI (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes (ANOVA; F (3, 8) = 9.374, p = 0.005, η 2 = 0.779), followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. NFI, nuclear fusion index.

Techniques: Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: BCAA reduced Atrogin-1 expression in DFO-treated myotubes, but had no effect on MuRF-1 expression. (A) Relative expression of Atrogin-1(F (3, 8) = 46.837, p < 0.001, η 2 = 0.946) (N = 3). (B) Relative expression of MuRF-1(F (3, 8) = 26.29, p < 0.001, η 2 = 0.908) (N = 3). (C) (D) Representative WB blot images showing the protein expression of Atrogin-1 and MuRF-1. Protein was extracted on 24 h after BCAA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments, and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Techniques: Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: BCAA increased the p-Akt in DFO-treated myotubes after 45 min of BCAA treatment, but had no statistically significant effects on other signaling molecules at the same time point. Representative blots for quantification of (A) p-Akt (F (3, 8) = 6.166, p = 0.018, η 2 = 0.6981), p-mTOR, p-p70S6K, p-4E-BP1, and p-eEF2, (B) p-AMPK, and p-ACC, and (C) p-FOXO1, and p–NF–κB p65. Protein was extracted at 45 min after BCCA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments (N = 3), and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05.

Techniques:

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: BCAA increased the phosphorylation of p70S6K in DFO-treated myotubes after 24 h of BCAA treatment, but had no effect on the phosphorylation of AMPK, Akt, or eEF2. (A) Representative blots for quantification of (B) p-AMPK(F (3, 8) = 5.620, p = 0.023, η 2 = 0.678), (C) p-Akt (F (3, 8) = 6.394, p = 0.016, η 2 = 0.706), (D) p-p70S6K(F (3, 8) = 16.43, p < 0.001, η 2 = 0.860), and (E) p-eEF2 (F (3, 8) = 10.23, p = 0.004, η 2 = 0.793). Protein was extracted 24 h after BCCA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments (N = 3), and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗p < 0.01.

Techniques: Phospho-proteomics

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myotubes (Tokyo Chemical Industry)

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