Journal: Aging Cell

Article Title: Secretome Profiling of Young Multipotent Stem Cells Reveals Angiogenic and Immunomodulatory Mechanisms Supporting Aged Neuromuscular Health

doi: 10.1111/acel.70408

Figure Lengend Snippet: Systemic transplantation of young MDSPCs promotes skeletal muscle neovascularization, improves muscle structure, and increases mitochondrial content. (A) Representative immunohistochemical images of gastrocnemius (GS) muscles from young MDSPC (NA‐CI; n = 9) and PBS (NA‐PBS; n = 7) treated mice, labeled for CD31 (red) and dystrophin (green). (B) Quantification of vasculature density, presented as the number of vessels per muscle fiber. (C) Quantification of total vasculature area, presented as percentage of muscle area occupied by vessels. (D) Volcano plot depicting statistical significance and fold change of neovascularization pathway protein phosphorylation levels in GS muscles from NA‐CI ( n = 4) and NA‐PBS ( n = 4) mice. Proteins that have sites with significantly increased phosphorylation in NA‐CI muscles are highlighted in green. (E) Representative images of GS muscles stained with Sirius red (collagen, red) and Fast Green (muscle, green) at 2 months post‐intraperitoneal (IP) transplantation. (F) Quantification of collagen content as a percentage of total tissue area. (G) Representative images of dystrophin‐labeled GS muscles (green) from NA‐CI and NA‐PBS mice. (H) Violin plot of muscle fiber cross‐sectional area (CSA), with lines indicating median and interquartile ranges (25th and 75th percentiles). (I) Frequency distribution of muscle fiber CSA binned in 100 μm 2 intervals. (J) Citrate synthase activity measured in quadriceps muscles of NA‐CI ( n = 9) and NA‐PBS ( n = 7) mice. (K) Representative images of GS muscles labeled for type I (blue), type IIa (green), and type IIb (red) muscle fibers at 2 months post‐IP transplantation. (L) Stacked bar plot of fiber type composition in GS muscles of NA‐CI ( n = 7) and NA‐PBS ( n = 7) mice. Data (B, C, F, I, J, L) are presented as mean ± SEM. ** p ≤ 0.01, *** p ≤ 0.001, and § p ≤ 0.0001 using one‐tailed unpaired Student's t ‐test. (H) § p ≤ 0.0001 by two‐tailed Kolmogorov–Smirnov test. Scale bars are 100 μm (A, G, K) and 500 μm (E).

Article Snippet: Muscle fiber cross‐sectional area was assessed by immunohistochemically (IHC) labeling dystrophin (Abcam, ab15277, 1:300), type I muscle fibers (DSHB, BA‐F8, 1:50), type IIa muscle fibers (DSHB, SC‐71, 1:600), type IIb muscle fibers (DSHB, BF‐F3, 1:100), and type IIx muscle fibers (DSHB, 6H1, 1:50), following established protocols (Vella et al. ; Lavasani et al. , ; Bloemberg and Quadrilatero ).

Techniques: Transplantation Assay, Immunohistochemical staining, Muscles, Labeling, Phospho-proteomics, Staining, Activity Assay, One-tailed Test, Two Tailed Test

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , weighted nearest neighbor (WNN) UMAP integrating snATAC-seq and snRNA-seq from tibialis anterior muscle 7 days post-denervation. Left panel colored by cluster identity shows major populations. Right panel colored by experimental group shows control (CTL, red) versus denervated (D, blue) b , Dot plot of marker gene expression across cell clusters. Y-axis lists 15 clusters; X-axis displays marker genes. Dot size indicates percent expression; color intensity (purple gradient) shows average expression level. Validates cluster identities through fiber type-specific Myh expression and cell type-specific markers. c , Coverage plots showing chromatin accessibility at f Myh locus for Myh4 /IIb, Myh2 /IIa, and Myh1 /IIx in control (CTL) and denervated (D) TA, with at the right violin plots showing the expression level of the corresponding gene. Below, in black each gene is represented. d , UMAP subset focusing on type IIb myonuclei. Left (CTL) shows control IIb nuclei right (D) shows resilient denervated IIb nuclei that retain spatial clustering with control population (D_IIb_res), suggesting maintenance of transcriptional/chromatin accessibility identity despite denervation. e , Coverage plots comparing Myh1 and Myh4 loci in resilient denervated population. Cyan tracks showing denervated nuclei; red tracks showing control nuclei. Violin plots (right) indicate gene expression. Myh1 and Myh4 genes are presented below corresponding the coverage plots. f , Venn diagram comparing differentially expressed genes between IIb denervated nuclei which cluster with the control (resilient myonuclei, 305 genes, orange) and those (non-resilient, 725 genes, green) that do not cluster with the control. Overlap shows 268 shared dysregulated genes; 37 unique to resilient, 457 unique to non-resilient.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Control, Marker, Gene Expression, Expressing

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , Dot plot showing expression of Maf family transcription factor, Runx1 , Hdac 4, Nr4a1 , Tbx15 , Musk , Nfe2l2 expression across distinct myonuclei populations. Y-axis lists seven cell populations. X-axis displays gene names. Dot size indicates percentage of cells expressing each gene (legend shows 0, 10, 20, 30, 40 percent). Dot color intensity (purple gradient) represents average expression level (scale 0 to 2 arbitrary units). Populations include control (IIa, IIx, IIb) and denervated (D_IIb, D_IIx) myonuclei plus specialized populations (NMJ, MTJ). b , Six UMAP feature plots displaying gene expression and chromatin accessibility patterns from WNN-integrated multiome dataset, as in Supplementary Figure 1a. Top row, left to right: c-Maf RNA expression (purple-to-gray gradient), half-MARE motif chromatin accessibility (brown-to-gray gradient), palindromic MARE motif accessibility (brown-to-gray gradient). Bottom row, left to right: Runx1 RNA expression (purple-to-gray gradient), RUNX1 binding motif accessibility (brown-to-gray gradient), FOS motif accessibility (brown-to-gray gradient). Color intensity indicates expression level or accessibility score per nucleus. c, Violin plots showing expression of type IIb fiber-specific genes in IIb myonuclei (red) versus denervated IIb myonuclei (D_IIb, cyan) from single-nucleus RNA-seq of tibialis anterior muscle at 7 days post-denervation. Denervation induces downregulation of the fast-glycolytic contractile program, including Myh4 (type IIb myosin heavy chain), Actn3 (alpha-actinin-3), and Mybpc2 (myosin binding protein C2), alongside metabolic enzymes Ckm (muscle creatine kinase) and Pvalb (Parvalbumin) and the sarcoplasmic reticulum calcium ATPase Atp2a1 (SERCA1). Each dot represents an individual myonucleus; violin width indicates cell density distribution. d , Chromvar analysis of MAF::NFE2 motif enrichment in NMJ compared to IIb CTL showing significant enrichment.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Expressing, Control, Gene Expression, RNA Expression, Binding Assay, RNA Sequencing

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , UMAP visualization of single-nucleus RNA-seq from plantaris muscle 7 days post-tenotomy (mechanical unloading) and sham-operated controls. Cells colored by cluster identity reveal 11 distinct populations: IIb (orange, type IIb/ Myh4 myonuclei), IIx (red, type IIx/ Myh1 myonuclei), FAPs (yellow, fibro-adipogenic progenitors), IIa (green, type IIa/ Myh2 myonuclei), Macrophages (teal), FAPS- Dpp4 + (cyan), Neuromuscular cells (blue), Tenocytes (light blue), Endothelial (purple), MuSC (magenta, muscle stem cells), Schwann cells (pink), MTJ (pink, myotendinous junction). Myonuclei clusters segregate by Myh isoform expression demonstrating fiber type-specific transcriptional states. b , Same UMAP colored by experimental condition using group.by function. Cyan indicates Ctrl-Pltr (control plantaris); pink indicates T-Pltr (tenotomized plantaris). Both conditions distribute across all clusters, enabling paired comparison of tenotomy effects within each cell type. c , Venn diagram analysis of Ingenuity Pathway Analysis (IPA) upstream regulators (URs). Left: Overlap between Tenotomy (57 unique URs) and Denervation (212 unique URs) showing 31 shared regulators, indicating partially overlapping stress responses. Right: Triple Venn diagram among the 212 unique denervation URs showing fiber type-specific ones: type IIa (82 unique), IIb (45 unique), and IIx (36 unique) myonuclei. Central overlaps show shared regulators: 5 (IIa/IIb), 12 (all three), 12 (IIb/IIx), and 20 (IIa/IIx). d , Table of IIb-specific upstream regulators showing z-scores and p-values across fiber types (IIa, IIb, IIx). Columns display regulator name, activation z-score, and statistical significance for each subtype. Red cells indicate activated pathways; blue indicates inhibited pathways. Red rectangles highlight two key regulators: KAT2B (Lysine acetyltransferase) showing its specific inhibition in IIb fibers, and HDAC5 (class IIa histone deacetylase) showing its specific activation. This inverse regulation suggests coordinated chromatin remodeling where HDAC5 activation and KAT2B inhibition converge to alter acetylation landscapes during denervation of fast glycolytic IIb myofibers.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: RNA Sequencing, Expressing, Control, Comparison, Activation Assay, Inhibition, Histone Deacetylase Assay

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , Ponceau S total protein staining serving as loading controls for western blots presented in main of the manuscript. Two membrane panels show protein transfer quality for soleus (SOL) and tibialis anterior (TA) muscles. Each panel displays cytoplasmic and nuclear protein fractions. b. Violin plots showing expression of c-Maf in Myh4 + myonuclei from single-nucleus RNA-seq in control plantaris and plantaris after tenotomy. c , UMAP visualization of single-nucleus RNA-seq from botulinum toxin A (BoTX)-injected muscles . Two Feature plots compare gene expression profiles: left panel shows BoTX_Ctrl (control-treated), right panel shows BoTX (toxin-treated). Each point represents an individual myonucleus positioned by UMAP dimensionality reduction. Color intensity (purple-to-gray gradient) indicates c-Maf expression level per nucleus, with “ Maf ” label on right margin. d, Widefield images at x10 magnification of Gastrocnemius muscle in CTL and after electrical stimulation (20 Hz and 100 Hz) stained for c-MAF (red), WGA (green) and Dapi (blue). e , Spinning-disk confocal images at x60 magnification of soleus muscle after electrical stimulation same protocol and staining as in d.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Staining, Western Blot, Membrane, Muscles, Expressing, RNA Sequencing, Control, Injection, Gene Expression

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , Immunofluorescence of distal hindlimb cryosections showing MYH isoform distribution and Laminin (white, fiber boundaries). Top rows: MYH1 (cyan, type IIx), MYH2 (green, type IIa), MYH7 (red, type I). Bottom rows: MYH4 (cyan, type IIb), MYH2 (green), MYH7 (red). Columns show: Control HSA-Cre + (leftmost), myofiber constitutive HSA-Cre;c-Maf flox/flox , myofiber inducible HSA-CreERT2;c-Maf flox/flox at 7 days, 15 days, and 30 days post-tamoxifen, Region A deleted (f Myh super enhancer deletion, EnhA -/- ), and ALS 90 days old. Major muscles labeled in control: TA (tibialis anterior), Tibia, EDL (extensor digitorum longus), FHL-TP (flexor hallucis longus-tibialis posterior), Sol (soleus), Plant (plantaris), Gas (gastrocnemius). c-Maf loss shows fiber type transition in peroneal group muscles (FHL-TP, central TA) with preserved EDL. ALS models displayed similar regional fiber type switch. b , RT-qPCR of fast-glycolytic genes in inducible c-Maf mutant 7 and 15 dpi of Tamoxifen. Bottom: Myh4 and Myh1 showing temporal changes. c-Maf loss reduces Myh4 expression while delayed Myh1 increase. Top shows Pvalb (parvalbumin), Atp2a1 (SERCA1), and Mybpc2 (fast MyBP-C) downregulation. White bars: control (CTL); pink bars: 7 dpi; red bars: 15 dpi. *p < 0.05 by one-way ANOVA with individual data points. c , RT-qPCR at 4 weeks post-tamoxifen comparing circadian timepoints ZT03 and ZT15. Left: Myh4 expression is reduced only at ZT03 (red bars) versus controls (white bars). Right: Myh1 upregulation in KO at ZT03. *p < 0.05, **p < 0.01 showing sustained fiber type shift. d , RT-qPCR on 90 days old ALS G93A mice comparing circadian timepoints ZT03 and ZT15. Top c-Maf expression sharply down regulated (grey bars) versus control (white bars). Middle left: Myh4 expression is constitutively reduced versus controls. Middle right: Myh1 is upregulated specifically at ZT03. Bottom left Myh4 expression only in 90 days old ALS mice is upregulated at ZT15. Bottom right same with Myh1. e, RNA-FISH on isolated TA fibers at ZT03 and ZT15, 4 weeks post-tamoxifen, targeting pre-mRNA of Myh4 (red), Myh1 (yellow), Myh2 (green). DAPI (blue) marks myonuclei. Yellow box shows high-magnification view of transcription foci shown in panel f . g, c-Maf mutant myofibers show increased myonuclei expressing non-dominant Myh isoforms, more pronounced at ZT03 than ZT15, indicating spatio-temporal discoordination of the fMyh locus. Violin plots, white color control, red c-Maf mutant. The % of heterogeneity is the % of f Myh allele that differs from the dominant f Myh allele per myofiber as estimated by Rnascope experiments. *p < 0.05, ***p < 0.001 ****p < 0.0001 by one-way ANOVA.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Immunofluorescence, Control, Muscles, Labeling, Quantitative RT-PCR, Mutagenesis, Expressing, Isolation, RNAscope

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a, Quantification of fiber type composition in tibialis anterior (TA) muscle from c-Maf mutant mice generated using constitutive HSA-Cre (Top) or inducible HSA-CreERT2 (Bottom), assessed by Myosin heavy chain (MYH) isoform immunostaining. Wild-type (black bars) and c- Maf knockout (white/gray bars) muscles show significant reduction in type IIb (MYH4) fiber content with compensatory increases in type IIx (MYH1) and type IIa (MYH2) fibers following c- Maf deletion. b, Cross-sectional area (CSA µm 2 ) measurements of individual fiber types in TA (top) and EDL (bottom) muscles from HSA-Cre (left) and HSA-CreERT2 (right) mutant mice. Selective atrophy of MYH4-expressing type IIb fibers is observed exclusively in TA muscle, with no CSA changes detected in EDL, revealing region-specific vulnerability to c-Maf loss despite comparable fiber type composition. Note that the CSA of MYH4 myofibers is around 3000 µm 2 in the TA, while of 1200 µm 2 in EDL.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Mutagenesis, Generated, Immunostaining, Knock-Out, Muscles, Expressing

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , Chip-seq alignment of c-MAF peaks with H3K27ac histone mark on Foxo3 gene showing c-MAF significant peak in intronic enhancer of Foxo3. b , Immunofluorescence of FOXO3 in flexor hallucis longus (FHL) and extensor digitorum longus (EDL) sections. MYH4 (cyan) and FOXO3A (red). c , Quantification of FOXO3-positive myonuclei across muscles (CTL-EDL, KO-EDL, CTL-TA, KO-TA, CTL-FHL-TP, KO-FHL-TP). **p < 0.01 Two-way ANOVA. d , Confocal imaging of isolated TA fiber nuclei showing FOXO3A (magenta) and DAPI (blue) at ZT03 and ZT15. e , RT-qPCR of atrophy genes Foxo3 , Nr3c1 , Nr3c2 , Fbxo32 , Foxo1 , Trim63 , Fbxo31 , Map1lc3a , Mstn , Fkbp5 ) at ZT03/ZT15. White bars (CTL), red bars (KO). *p < 0.05, **p < 0.01 One-way ANOVA. f , RT-qPCR of protein synthesis genes ( Castor2 , Eif4ebp1 , Slc7a2 , Eif4e ). g , Gene expression kinetics after c-Maf deletion. Graphs showing Foxo3 , Fbxo32 , Trim63 expression at 7 and 15 dpi of tamoxifen, *p < 0.05 One-way ANOVA. h , Expression of Castor2 , Eif4ebp1 and Slc7a2 in 90-day old CTL and ALS SOD1-G93A at ZT03/ZT15. ALS and c-Maf KO mice showed comparable dysregulation of those genes. *p < 0.05, **p < 0.01 One-way ANOVA.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: ChIP-sequencing, Immunofluorescence, Muscles, Imaging, Isolation, Quantitative RT-PCR, Gene Expression, Expressing

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , Immunofluorescence of distal hindlimb cryosections comparing muscle-specific large Maf family mutant models ( HSA-Cre;c-Maf flox/flox , HSA-Cre;Mafb flox/flox and Mafa null mice). Four panels showing transverse sections from: wild-type (WT), c-Maf single mutant, Mafa single mutant, and Mafb single mutant mice. Myosin heavy chain (MYH) isoforms visualized by immunostaining: MYH7 (blue, slow oxidative), MYH2 (red, fast oxidative), MYH1 (white, fast oxidative-glycolytic), MYH4 (green, fast glycolytic), and Laminin (magenta, basal lamina marking fiber boundaries). White arrows indicating flexor hallucis longus (FHL) and tibialis posterior (TP) muscles showing region-specific fiber type transition in c-Maf mutant. b , RT-qPCR quantification of genes specifically dysregulated in c-Maf mutant compared to Mafb and Mafa mutants. Seven bar graphs show relative mRNA expression for: Klk1b26 , Prkaa2 , Fbxo32 , Foxo3 , Fkbp5 , Fbxo31 , and Eif4e . X-axis categories: CTL (control), total Mafa mutant, muscle specific Mafb mutant, muscle specific c-Maf mutant . Statistical comparisons by one-way ANOVA with post-hoc testing. Asterisks indicate significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Immunofluorescence, Mutagenesis, Immunostaining, Muscles, Quantitative RT-PCR, Expressing, Control

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a, Low-magnification (×20) confocal images of RNA-FISH targeting Myh4 mRNA (green) and Etv5 mRNA (red) in isolated TA fibers 4-6 weeks post-tamoxifen injection in HSA- CreERT2;c-Maf flox/flox . Left: Control (CTL) showing abundant Etv5 expression at NMJ (white arrows). Right: c-Maf mutant displayed markedly reduced Etv5 signal. NMJ regions are indicated by white arrows. b , High-magnification (×60) images of c-Maf mutant myofibers showing two patterns. Top row: fiber with suspected NMJ location exhibiting almost complete loss of Etv5 mRNA at NMJ. Bottom row: fiber displaying dual expression pattern with Myh4 mRNA (green), Etv5 mRNA (red), and merge. c, Experimental timeline and schematic design. HSA-CreERT2;c-Maf flox/flox mice received three tamoxifen injections (×3) to induce c-Maf deletion, followed by treatment with α-bungarotoxin-488 at 5 days and α-bungarotoxin-555 10 days post-injection to assess AchR turn-over in TA muscles. d, Confocal immunofluorescence images of NMJs from control (CTL) and mutant (KO) mice, showing distribution of old acetylcholine receptors (AchR; green) and newly synthesized AchR (red). Representative merged images show overlay of old and new AchR signals. e, Quantification of relative AchR turnover in CTL versus mutant mice. Mutant mice show significantly elevated AchR turnover compared to CTL (*** P < 0.001, unpaired t-test). f, Quantification of mean AchR fragments per NMJ in CTL and mutant mice. g, Confocal immunofluorescence images of NMJs from CTL and mutant mice at circadian timepoints ZT03 and ZT15, showing α-bungarotoxin labeling (red), FOS (green), and FOXO3 (magenta). The merged channel reveals colocalization patterns and temporal dynamics of marker expression. Notably, mutant mice display increased FOXO3 signal and altered FOS/α-bungarotoxin overlap patterns compared to CTL.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Isolation, Injection, Control, Expressing, Mutagenesis, Muscles, Immunofluorescence, Synthesized, Labeling, Marker

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , Representative fluorescence in situ hybridization images showing Myh4 (green), Etv5 (red), and Fos (magenta) mRNA expression with DAPI nuclear counterstain (blue) in TA isolated fibers of c-Maf inducible mutant 4 weeks after tamoxifen injection at Zeitgeber time 3 (ZT03) and Zeitgeber time 15 (ZT15). b , Quantification of Etv5 mRNA puncta volume at NMJ showing significant reduction in mutant animals at both ZT03 and ZT15 compared to CTL (***P < 0.001), and a trend toward augmentation in KO ZT15 versus KO ZT03 (P = 0.06). Each dot represents a single measurement; horizontal lines indicate mean values. Statistical test: Two-way ANOVA. c , Quantification of Fos mRNA puncta volume at the NMJ across genotypes and Zeitgeber times. No significant differences were observed between groups. Each dot represents a single measurement; horizontal lines indicate mean values. d, Quantification of acetylcholine receptor (AChR) fragmentation at neuromuscular junctions in control (CTL), 7 days post-tamoxifen injection (7dpi), and 15 days post-tamoxifen injection (15dpi) in HSA-CreERT2; c-Maf mutant tibialis anterior muscle. Progressive NMJ fragmentation occurs following c-Maf deletion, with significant increases in mean AChR fragment number by 15 days post-tamoxifen. e, Representative confocal z-stack maximum projection images of neuromuscular junctions at 4 weeks post-tamoxifen injection showing co-localization of α-bungarotoxin (α-BTX, red) labeling postsynaptic AChRs, c-FOS (green), and FOXO3A (magenta) indicating activation of atrophy-related transcriptional programs. Merge image shows nuclear DAPI (blue) with overlay of all channels, demonstrating nuclear accumulation of FOXO3A in myonuclei associated with fragmenting neuromuscular junctions following c-Maf deletion.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Fluorescence, In Situ Hybridization, Expressing, Isolation, Mutagenesis, Injection, Control, Labeling, Activation Assay