c2c12 cells  (ATCC)

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

Journal: Journal of Cellular Physiology

Article Title: PIEZO1 Channels Modulate the Small Extracellular Vesicle Release in C2C12 Cells

doi: 10.1002/jcp.70155

Figure Lengend Snippet: Yoda1‐induced sEV promotes myotube formation. (A) Representative optical field areas of 4‐day differentiating C2C12 cells in control conditions (Ctrl; DM medium), treated with exogenous sEV derived from MB and MT cultures (cMB‐sEV and cMT‐sEV) or with exogenous sEV derived from Yoda1‐treated MB and MT cultures (yMB‐sEV and yMT‐sEV). Fluorescence images (DAPI staining) were overlaid on the corresponding bright‐field images. (B) The number of myotubes in each optical field (OF) was normalized to the average number of myotubes observed in Ctrl condition. For each experimental point, at least 40 randomly selected.

Article Snippet: Myoblasts derived from the C2C12 cell line (RRID: CVCL_0188, ATCC‐CRL‐1772, LCG Standards S.r.l, MI, Italy) were kept in growth media (GM) containing Dulbecco's Modified Eagle Medium‐High Glucose (DMEM, Sigma‐Aldrich, St. Louis, Missouri, USA), 20% Fetal Bovin Serum (FBS; EuroClone, Pero, MI, Italy), 4 mM l ‐Glutamine (EuroClone, Perom MI, Italy), 1% Penicillin‐Streptomycin (EuroClone, Pero, MI, Italy), at 37°C in humidified air containing 5% CO 2 .

Techniques: Control, Derivative Assay, Fluorescence, Staining