Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Comparative analysis of conventional and brush-assisted bioprinting on cellular behavior. (a) Schematic illustration of shear stress distribution in normal versus brush-assisted printing. (b) Overview of the brush-assisted printing setup. (c) SEM images of collagen fibrils and fluorescence images showing cell viability (live/dead) and cytoskeletal organization (DAPI/phalloidin) of C2C12 myoblasts. Quantification of (d) cell viability post-printing (n = 4), (e) cell metabolic activity (MTT assay, in situ /day 3/day 7, n = 4), (f) nuclei aspect ratio (n = 180), (g) orientation factor (n = 3), and (h) F-actin–positive area at day 3 (n = 10). (i) Comparing normal and brush-assisted printing the mechanotransduction pathways activated by shear stress and collagen alignment. (j) Heatmap of relative gene expression ( YAP, TAZ, AKT1, PIEZO1, PI3K, and CAPN2 ) after 7 days of culture (n = 4). (k) Agarose gel electrophoresis of PCR products from cells cultured on normal versus brush-printed scaffolds for 7 days. (l) Schematic illustration of blocking mechano-sensing ion channel with GsMTx-4. (m) Relative gene expression levels associated with mechanosensing channel and ca 2+ pathway (n = 5), (n) Hippo pathway (n = 5), (o) PI3K-AKT pathway (n = 5). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (cell metabolic activity assay); F-actin, Filamentous actin; PI3K , Phosphoinositide 3-kinase; CAPN2 , Calcium-activated neutral protease 2.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Shear, Fluorescence, Activity Assay, MTT Assay, In Situ, Gene Expression, Agarose Gel Electrophoresis, Cell Culture, Blocking Assay, Standard Deviation, Metabolic Assay

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Physical and biological evaluation of bioconstructs reinforced with straight (CSP) and coiled (CCP) PCL fibers. (a) Schematic illustration and optical images of flow tests with Col (collagen-only), CSP, and CCP bioinks, in which droplets were tilted at 90° for 10 min. (b) Quantification of droplet outflow (n = 10). Rheological measurements of bioinks: (c) storage modulus (G′) from frequency sweep (n = 3), (d) G′ and G″ from temperature sweep (n = 3), and (e) G′ and G″ under cyclic stress loading (10 and 200 Pa) showing viscoelastic recovery (n = 3). (f) SEM images of CCP constructs highlighting coiled PCL fiber and aligned collagen fibrils. (g) Optical image, live/dead staining, DAPI/phalloidin staining, and orientation factor analysis of C2C12 cells and PCL microfibers. Quantification of (h) cell viability (live/dead, n = 4), (i) F-actin–positive area (n = 20), and (j) metabolic activity (MTT assay, in situ /day 3/day 7, n = 4). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗).

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Construct, Staining, Activity Assay, MTT Assay, In Situ, Standard Deviation

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: In vitro myogenic differentiation of C2C12 cells cultured on Col, CSP and CCP scaffolds. (a) Live/dead staining at in situ , DAPI/phalloidin staining at day 3, and DAPI/ MHC staining at day 14. (b) Quantification of cell viability from live/dead assays (n = 4). (c) Nuclei orientation factor after 7 days of culture (n = 4). (d) Nuclei aspect ratio (n = 20). (e) F-actin positive area (n = 4). (f) Quantification of MHC fusion index (left, n = 5) and MHC maturation rate (right, n = 5). (g) Relative gene expression analysis and (h) agarose gel electrophoresis regarding mechanotransduction-related genes ( CAPN2, PIEZO1, RhoA, YAP, and TAZ ) (n = 4). (i) Western blot analysis of PIEZO1 . (j) Schematic illustrating differentiation progression and major genes involved at each stage. (k) Heatmap and (l) agarose gel electrophoresis of PCR products showing relative expression of myogenic markers ( MYF5, MYOD1, MYOG, MHC, MYH2, and MYH4 ) after 21 days of culture (n = 4). (m) Western blot analysis of MHC . Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MYF5 , Myogenic factor 5; MYOD1 , Myogenic differentiation 1; MYOG , Myogenin; MHC , Myosin heavy chain; MYH2 , Myosin heavy chain 2; MYH4 , Myosin heavy chain 4.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: In Vitro, Cell Characterization, Cell Culture, Staining, In Situ, Gene Expression, Agarose Gel Electrophoresis, Western Blot, Expressing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Attenuated lamin A–prohibitin2 interaction leads to mitochondrial dysfunction in LMNA 289 A>G–mediated dilated cardiomyopathy

doi: 10.1016/j.jbc.2026.111274

Figure Lengend Snippet: Loss of interaction with PHB2 complex in lamin A mutant K97E. A , schematic representation of the K97E mutation in the 1B domain of lamin A (not to scale). B , schematic overview of proximity-dependent biotinylation and streptavidin pulldown (BioID2) strategy. C , Z-projected immunofluorescence images showing localization of myc-BioID2-lamin A variants (the scale bar represents 2 μm). D , intensity profiles across the nuclear axis depicting myc-BioID2-lamin A and overall lamin A distribution. E , Pearson's correlation coefficient analysis showing colocalization between myc and lamin A signals. F , immunoblot of lamin A/C and GAPDH showing equal BioID2 input loading and comparable expression of BioID2-lamin A variants in HEK293T cells. G , number of interacting partners of WT and K97E lamin A as observed from protein identification through BioID2-pulldown followed by MS. H , Venn diagram indicating independent and overlapping interactome of WT and K97E lamin A. I , enrichment analysis of protein–protein interactions lost in the K97E mutant compared with WT lamin A. J , BioID2-based immunoblot analysis showing reduced interaction between lamin A and PHB2 in the K97E mutant compared with WT. K , quantitative PCR (qPCR) analysis of PHB1 and PHB2 mRNA levels, revealing differential expression in response to the K97E mutation in C2C12 cells. L , immunoblot analysis of PHB1, PHB2, GAPDH, and lamin A in C2C12 cells. M , densitometric quantification of PHB1 and PHB2 immunoblots, normalized to GAPDH. N , Z-projected immunofluorescence images of PHB2 showing altered subcellular distribution upon K97E lamin A expression (the scale bar represents 4 μm). O , quantitative analysis of PHB2 fluorescence intensity from immunofluorescence imaging. P , subcellular fractionation of cells expressing WT and K97E lamin A variants, followed by immunoblot against GAPDH (cytosolic marker), lamin A (nuclear marker), and PHB2. Q , densitometric quantification of PHB2 levels in the cytoplasmic fraction, normalized to GAPDH. R , in silico molecular docking of PHB2 with lamin A variants highlighting lamin A LYS97 and PHB2 GLU231 as potential perturbed interaction. S , blot overlay of purified lamin A proteins overlayed with whole cell lysate and probed with PHB2 to detect interaction. T , far Western of purified lamin A proteins with cell lysate and probed with PHB2 to detect interaction. HEK293T, human embryonic kidney 293T cell line; PHB2, prohibitin 2.

Article Snippet: HEK293T (ATCC) cells were maintained using the same parameters as C2C12.

Techniques: Mutagenesis, Immunofluorescence, Western Blot, Expressing, Protein-Protein interactions, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Fluorescence, Imaging, Fractionation, Marker, In Silico, Purification

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Biochemical and live-cell imaging characterization of the effects of insulin on INSR. (A) Internalized to total INSR ratio was quantified using surface biotinylation in undifferentiated C2C12 myoblasts. Cells were incubated in PBS (no insulin) or serum-free DMEM containing 0, 0.2, 2, or 20 nM insulin for 15 minutes. The ratios are normalized to the 0 nM group of each gel ( P > .05 when not specified). (B) Western blot showing SNAP-tagged INSR expressed from lentiviral vector in comparison to wild-type INSR in C2C12 myoblasts. (C, D) Representative images of INSR-A-SNAP-labeled using an Alexa Fluor 488 cell nonpermeable dye in 0, 0.2, or 20 nM insulin conditions in live undifferentiated C2C12 myoblasts from (C) 0 to 15 minutes or (D) after 20 minutes using a spinning disk confocal microscope. Time-lapse images of INSR vesicle interactions starting from the selected subregions (white squares) of snapshots are shown in the insets ( n = 3 cells).

Article Snippet: The C2C12 mouse myoblast (ATCC cell line provided by Dr Brian Rodrigues, University of British Columbia, Vancouver, Canada) was maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), and 1% penicillin–streptomycin (100 μg/mL; Gibco).

Techniques: Live Cell Imaging, Incubation, Western Blot, Plasmid Preparation, Comparison, Labeling, Microscopy

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Interaction and colocalization between INSR, CAV3, CAV1, and CLTC under different insulin stimulations. (A) Western blot of mice skeletal muscle lysates after insulin injection. Phospho-AKT (Ser473) to total AKT ratio or phospho-ERK1/2 to total ERK ratio was quantified ( n = 6). (B) Co-immunoprecipitation of skeletal muscle INSR after PBS or insulin injection. CLTC to INSR ratio or CAV3 to INSR ratio were quantified ( n = 7-10). (C) Representative STED microscopy images of C2C12 myoblasts expressing INSR-A-SNAP (surface labeled) that were fixed after stimulation with 0, 0.2, or 20 nM insulin for 30 minutes and stained for CAV1 and CLTC (scale bar = 5 µm). (D, E) Colocalizations between INSR-A-SNAP, CAV1, and CLTC were quantified by Object Pearson Coefficient. Data are plotted to show differences between insulin concentrations (D) or between protein pairs (E) ( n = 7-9 images, 1-4 cells per image. * P < .05, Tukey's multiple comparison after 2-ANOVA. Box represents median and 25th to 75th percentiles).

Article Snippet: The C2C12 mouse myoblast (ATCC cell line provided by Dr Brian Rodrigues, University of British Columbia, Vancouver, Canada) was maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), and 1% penicillin–streptomycin (100 μg/mL; Gibco).

Techniques: Western Blot, Injection, Immunoprecipitation, Microscopy, Expressing, Labeling, Staining, Comparison

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Colocalization and interaction between INSR and ANXA2 under different insulin concentrations. (A, B) Representative STED microscopy images of C2C12 myoblasts expressing INSR-A-SNAP (surface labeled) that were fixed after stimulation with 0, 0.2, or 20 nM insulin for (A) 15 or (B) 30 minutes and stained for ANXA2 (scale bar = 5 µm). (C, D) Colocalization between INSR-A-SNAP and ANXA2 at (C) 15 or (D) 30 minutes of insulin-stimulated were quantified by Object Pearson Coefficient ( n = 4-13 images, 1-3 cells per image. # P < .05, 1-ANOVA of all groups. Box represents median and 25th to 75th percentiles.).

Article Snippet: The C2C12 mouse myoblast (ATCC cell line provided by Dr Brian Rodrigues, University of British Columbia, Vancouver, Canada) was maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), and 1% penicillin–streptomycin (100 μg/mL; Gibco).

Techniques: Microscopy, Expressing, Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: Attenuated lamin A–prohibitin2 interaction leads to mitochondrial dysfunction in LMNA 289 A>G–mediated dilated cardiomyopathy

doi: 10.1016/j.jbc.2026.111274

Figure Lengend Snippet: Loss of interaction with PHB2 complex in lamin A mutant K97E. A , schematic representation of the K97E mutation in the 1B domain of lamin A (not to scale). B , schematic overview of proximity-dependent biotinylation and streptavidin pulldown (BioID2) strategy. C , Z-projected immunofluorescence images showing localization of myc-BioID2-lamin A variants (the scale bar represents 2 μm). D , intensity profiles across the nuclear axis depicting myc-BioID2-lamin A and overall lamin A distribution. E , Pearson's correlation coefficient analysis showing colocalization between myc and lamin A signals. F , immunoblot of lamin A/C and GAPDH showing equal BioID2 input loading and comparable expression of BioID2-lamin A variants in HEK293T cells. G , number of interacting partners of WT and K97E lamin A as observed from protein identification through BioID2-pulldown followed by MS. H , Venn diagram indicating independent and overlapping interactome of WT and K97E lamin A. I , enrichment analysis of protein–protein interactions lost in the K97E mutant compared with WT lamin A. J , BioID2-based immunoblot analysis showing reduced interaction between lamin A and PHB2 in the K97E mutant compared with WT. K , quantitative PCR (qPCR) analysis of PHB1 and PHB2 mRNA levels, revealing differential expression in response to the K97E mutation in C2C12 cells. L , immunoblot analysis of PHB1, PHB2, GAPDH, and lamin A in C2C12 cells. M , densitometric quantification of PHB1 and PHB2 immunoblots, normalized to GAPDH. N , Z-projected immunofluorescence images of PHB2 showing altered subcellular distribution upon K97E lamin A expression (the scale bar represents 4 μm). O , quantitative analysis of PHB2 fluorescence intensity from immunofluorescence imaging. P , subcellular fractionation of cells expressing WT and K97E lamin A variants, followed by immunoblot against GAPDH (cytosolic marker), lamin A (nuclear marker), and PHB2. Q , densitometric quantification of PHB2 levels in the cytoplasmic fraction, normalized to GAPDH. R , in silico molecular docking of PHB2 with lamin A variants highlighting lamin A LYS97 and PHB2 GLU231 as potential perturbed interaction. S , blot overlay of purified lamin A proteins overlayed with whole cell lysate and probed with PHB2 to detect interaction. T , far Western of purified lamin A proteins with cell lysate and probed with PHB2 to detect interaction. HEK293T, human embryonic kidney 293T cell line; PHB2, prohibitin 2.

Article Snippet: HEK293T (ATCC) cells were maintained using the same parameters as C2C12.

Techniques: Mutagenesis, Immunofluorescence, Western Blot, Expressing, Protein-Protein interactions, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Fluorescence, Imaging, Fractionation, Marker, In Silico, Purification

Journal: The Journal of Biological Chemistry

Article Title: Attenuated lamin A–prohibitin2 interaction leads to mitochondrial dysfunction in LMNA 289 A>G–mediated dilated cardiomyopathy

doi: 10.1016/j.jbc.2026.111274

Figure Lengend Snippet: Loss of interaction with PHB2 complex in lamin A mutant K97E. A , schematic representation of the K97E mutation in the 1B domain of lamin A (not to scale). B , schematic overview of proximity-dependent biotinylation and streptavidin pulldown (BioID2) strategy. C , Z-projected immunofluorescence images showing localization of myc-BioID2-lamin A variants (the scale bar represents 2 μm). D , intensity profiles across the nuclear axis depicting myc-BioID2-lamin A and overall lamin A distribution. E , Pearson's correlation coefficient analysis showing colocalization between myc and lamin A signals. F , immunoblot of lamin A/C and GAPDH showing equal BioID2 input loading and comparable expression of BioID2-lamin A variants in HEK293T cells. G , number of interacting partners of WT and K97E lamin A as observed from protein identification through BioID2-pulldown followed by MS. H , Venn diagram indicating independent and overlapping interactome of WT and K97E lamin A. I , enrichment analysis of protein–protein interactions lost in the K97E mutant compared with WT lamin A. J , BioID2-based immunoblot analysis showing reduced interaction between lamin A and PHB2 in the K97E mutant compared with WT. K , quantitative PCR (qPCR) analysis of PHB1 and PHB2 mRNA levels, revealing differential expression in response to the K97E mutation in C2C12 cells. L , immunoblot analysis of PHB1, PHB2, GAPDH, and lamin A in C2C12 cells. M , densitometric quantification of PHB1 and PHB2 immunoblots, normalized to GAPDH. N , Z-projected immunofluorescence images of PHB2 showing altered subcellular distribution upon K97E lamin A expression (the scale bar represents 4 μm). O , quantitative analysis of PHB2 fluorescence intensity from immunofluorescence imaging. P , subcellular fractionation of cells expressing WT and K97E lamin A variants, followed by immunoblot against GAPDH (cytosolic marker), lamin A (nuclear marker), and PHB2. Q , densitometric quantification of PHB2 levels in the cytoplasmic fraction, normalized to GAPDH. R , in silico molecular docking of PHB2 with lamin A variants highlighting lamin A LYS97 and PHB2 GLU231 as potential perturbed interaction. S , blot overlay of purified lamin A proteins overlayed with whole cell lysate and probed with PHB2 to detect interaction. T , far Western of purified lamin A proteins with cell lysate and probed with PHB2 to detect interaction. HEK293T, human embryonic kidney 293T cell line; PHB2, prohibitin 2.

Article Snippet: C2C12 cells (American Type Culture Collection [ATCC]) were cultured in ATCC-formulated Dulbecco's modified Eagle's medium supplemented with penicillin–streptomycin and Glutamax (Gibco) as previously described ( , , ).

Techniques: Mutagenesis, Immunofluorescence, Western Blot, Expressing, Protein-Protein interactions, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Fluorescence, Imaging, Fractionation, Marker, In Silico, Purification

Journal: Nucleic Acids Research

Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

doi: 10.1093/nar/gkag168

Figure Lengend Snippet: CU-rich RNA promotes heterochromatin condensate organization during differentiation. ( A ) Nuclei of C2C12 myotubes (MT) treated with 1.5% 1,6-hexanediol (1,6-HD) or 1.5% 2,5-hexanediol (2,5-HD). Left: Representative images of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar, 5 μm. Middle: Time-course quantification of the number of heterochromatin foci per nucleus. Right: Boxplots show foci area (μm 2 , y -axis) at 5, 10, and 15 min posttreatment ( x -axis). n = 55 nuclei, three biological replicates. ( B ) Representative live-cell images of Hoechst 33342-stained MT nuclei before and after 1,6-HD treatment (0 and 15 min, respectively), taken from . Arrows indicate changes in heterochromatin foci intensity (pink, increased; blue, decreased), and the red arrow highlights an alteration in chromocenter integrity. ( C ) Number of heterochromatin foci per nucleus in MT following recovery from 1.5% 1,6-hexanediol (1,6-HD) treatment for 15 min, measured at indicated time points. n = 68 nuclei, three biological replicates. ( D ) Representative images of nuclei of myoblast (MB) and MT with or without 1,6-HD treatment (1.5%, 15 min). Right: Quantification of the number of heterochromatin foci per nucleus. n = 40 (MB), n = 60 (MT), three biological replicates. ( E ) Quantification of colocalization between indicated proteins and DAPI foci in MT with or without 1,6-HD treatment (1.5%, 15 min), by Pearson’s correlation coefficients. n = 60, three biological replicates. ( F ) Boxplot showing the distribution of Z -score of the interchromosomal interaction frequencies in MB and MT. ( G ) RNA FISH using ChRO1 and LacZ biotinylated probes. Biotin signal was detected by Fluorescein-conjugated Avidin DCS and amplified with biotinylated anti-Avidin and additional Fluorescein Avidin DCS. Right: Quantification of colocalization between biotin signal and DAPI foci. n = 50 nuclei. ( H ) Number of heterochromatin foci per nucleus of mouse fibroblast cells (NIH3T3) with or without doxycyline (Dox)-induced ChRO1a expression and/or 1,6-HD treatment (1.5%, 15 min). (EV; empty vector). n = 50, three biological replicates. ( I ) Number of heterochromatin foci per nucleus in MB with or without Dox-induced ChRO1a fragment (1–413, CUR) expression and/or 1,6-HD treatment (1.5%, 15 min). n = 75, three biological replicates. Statistical analyses and data presentation details are described in the “Materials and methods” section.

Article Snippet: C2C12 murine myoblast cells and NIH3T3 mouse fibroblast cells were obtained from the American-type culture collection and grown in a growth medium (GM) consisting of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Staining, Avidin-Biotin Assay, Amplification, Expressing, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

doi: 10.1093/nar/gkag168

Figure Lengend Snippet: ChRO1 deficiency and condensate disruption induce a muscle atrophic phenotype. ( A ) Strategy for generating ChRO1 KO (ChRO1−/−) mice using CRISPR–Cas9 genome editing targeting two gRNA sites to delete ChRO1 promoter, exon1, and part of intron1 region. ( B ) qRT-RCR quantification of ChRO1a and ChRO1b expression in gastrocnemius from 13-months-old-male mice WT and KO mice (ChRO1+/+ and ChRO1−/−). ( C ) qRT-PCR analysis of atrophic genes and satellite RNAs in gastrocnemius muscle from WT and ChRO1 KO mice. ( D ) Representative wheat germ agglutinin staining (left) and myofiber CSA (μm 2 ) quantification (right) of indicated skeletal muscle tissues from WT and ChRO1 KO mice. n ≥ 500 fibers analyzed per tissue. Scale bars, 40 μm. ( E ) Representative images (left) and quantification (right) of heterochromatin foci in gastrocnemius muscle from WT and ChRO1 KO mice. Scale bars, 1 μm. n = 50 nuclei. ( F ) Immunostaining for DAXX and H3K9me3 in gastrocnemius muscle of WT and ChRO1 KO mice. n = 50 nuclei. Scale bars, 2 μm. ( G ) qRT-PCR analysis of Atrogin1 (left) and Murf1 (right) in C2C12 MT differentiated for 5 days followed by 1,6-HD treatment at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Expression normalized to Gapdh and shown as fold change relative to untreated control for each time point. ( H ) Western blot analysis of MyHC protein levels in C2C12 MT treated with 1,6-HD at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Immunofluorescence staining of MyHC (AF488, green) and nuclei (DAPI, blue) ( I ) and quantification of myotube diameters [( J ), n = 100 cells] of C2C12 MT treated with 1,6-HD at indicated concentrations. ( K ) Quantification of myotube diameters of C2C12 MT treated with 2,5-HD or 1,6-HD at indicated concentration for 24 h. n = 45 cells. Statistical analyses, box plot elements, and data normalization procedures are detailed in the “Materials and methods” section.

Article Snippet: C2C12 murine myoblast cells and NIH3T3 mouse fibroblast cells were obtained from the American-type culture collection and grown in a growth medium (GM) consisting of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Disruption, CRISPR, Expressing, Quantitative RT-PCR, Staining, Immunostaining, Concentration Assay, Control, Western Blot, Immunofluorescence