Journal: bioRxiv

Article Title: Functionally Mature Bioengineered Human Skeletal Muscle Tissues Capture Essential Aspects of Glucose Metabolism

doi: 10.64898/2026.01.16.698404

Figure Lengend Snippet: (A) RNA-seq expression (counts per million, CPM) of canonical myogenic regulators (MYF5, MYF6, MYOD1, MYOG) across the 3D tissue maturation time course (days 7, 14, 21, 28, and 35). (B) Quantification of MYOD and myogenin protein abundance across maturation (days 2, 7, 16, and 23) from immunoblots in D. (C) RNA-seq expression (CPM) of metabolic maturation markers grouped by pathway: glucose metabolism (GYS1, HK2, PFKM), mitochondria/oxidative phosphorylation (ATP5F1A, CS, NDUFB8, SDHB, TFAM, UQCRC2), and AMPK subunit isoforms (PRKAA1/2, PRKAB1/2, PRKAG1/3) across the 3D maturation time course (days 7–35). (D) Representative western blots across maturation (days 2, 7, 16, and 23) for myogenic regulators (MYOD, myogenin), contractile/transport proteins (SERCA2), mitochondrial/OXPHOS proteins (NDUFB8, SDHB, UQCRC2, COX-II, ATP5A, CS), glycolysis (PFK1, HKII), glycogen metabolism (GS), and AMPK subunit isoforms (α1, α2, β1, β2, γ1, γ3). Coomassie staining is shown as a loading control. Bottom panel shows myosin heavy chain isoforms (MHC I, MHC IIa, MHC IIx). Statistical comparisons were performed using one-way ANOVA with appropriate multiple-comparisons correction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following antibodies were used: SERCA2 (SCBT sc-53010), Hexokinase II (SCBT sc-130358), Glycogen synthase (was a kind gift from Oluf Pedersen, University of Copenhagen, DK), Phosphofructokinase (SCBT sc-166722), Mito cocktail (NDUFB8, SDHB, UQCRC2, COX-II, ATP5A) (Abcam ab110411), Citrate Synthase (Abcam ab96600), AMPK α 1 (a kind gift from Olga Göransson, Lund University, SE), α 2 (MRC PPU Reagents and Services, University of Dundee, Scotland, UK, custom made), β 1 (SCBT sc-100357), β 2 (1.5, a kind gift from Dr. DG Hardie, University of Dundee, Scotland, UK), γ 1 (Abcam ab32508), γ 3 (Genscript, NJ, USA, custom made), Myogenin (Abcam ab1835), MyoD1 (Thermo Scientific, Waltham, MA, PA5-23078), pHSL Ser660 (CST #4126), pCREB Ser133 (CST #9198), pRPS6 Ser 236/236 (CST #2211), pRPS6 Ser240/244 (CST #2215), GLUT4 (Thermo Scientific, Waltham, MA, PA1-1065), PDH-E1α (SC-377092).

Techniques: RNA Sequencing, Expressing, Quantitative Proteomics, Western Blot, Phospho-proteomics, Staining, Control

Journal: bioRxiv

Article Title: Functionally Mature Bioengineered Human Skeletal Muscle Tissues Capture Essential Aspects of Glucose Metabolism

doi: 10.64898/2026.01.16.698404

Figure Lengend Snippet: (A) Heatmaps showing expression of slow-twitch, fast-twitch, general skeletal muscle, and non-skeletal muscle gene sets in bioengineered tissues at day 7 and day 21 compared with 7-day 2D monolayer myotubes. (B) SERCA2 protein levels measured across differentiation time points. (C) Protein abundance and relative proportion of myosin heavy chain isoforms. (D) Protein abundance of mitochondrial electron transport chain subunits from complexes I–V, including NDUFB8, SDHB, UQCRC1, COX4, ATP5A, and citrate synthase (CS). (E) AiryScan confocal images of COX4 staining (green) and actin (grey) in muscle tissues. (F) Quantification of metabolic enzymes including hexokinase II, glycogen phosphorylase, and phosphorylase kinase at indicated time points. (G) Protein expression of glycolytic enzymes and AMPK subunits, including AMPKα2, AMPKβ2, AMPKγ1, and AMPKγ3. Corresponding gene-expression data and representative western blots are shown in . Data (n=3) are presented as mean ± SEM, with individual values shown where applicable. Statistical comparisons were performed using one-way or two-way ANOVA with appropriate multiple-comparisons correction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. EMT = Bioengineered human muscle tissues, Monolayer = myotubes cultured in monolayer. Scale Bar =5 mm.

Article Snippet: The following antibodies were used: SERCA2 (SCBT sc-53010), Hexokinase II (SCBT sc-130358), Glycogen synthase (was a kind gift from Oluf Pedersen, University of Copenhagen, DK), Phosphofructokinase (SCBT sc-166722), Mito cocktail (NDUFB8, SDHB, UQCRC2, COX-II, ATP5A) (Abcam ab110411), Citrate Synthase (Abcam ab96600), AMPK α 1 (a kind gift from Olga Göransson, Lund University, SE), α 2 (MRC PPU Reagents and Services, University of Dundee, Scotland, UK, custom made), β 1 (SCBT sc-100357), β 2 (1.5, a kind gift from Dr. DG Hardie, University of Dundee, Scotland, UK), γ 1 (Abcam ab32508), γ 3 (Genscript, NJ, USA, custom made), Myogenin (Abcam ab1835), MyoD1 (Thermo Scientific, Waltham, MA, PA5-23078), pHSL Ser660 (CST #4126), pCREB Ser133 (CST #9198), pRPS6 Ser 236/236 (CST #2211), pRPS6 Ser240/244 (CST #2215), GLUT4 (Thermo Scientific, Waltham, MA, PA1-1065), PDH-E1α (SC-377092).

Techniques: Expressing, Quantitative Proteomics, Staining, Gene Expression, Western Blot, Cell Culture