Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: (A) Workflow for isolating fresh SCs (I) and single fibers (II) from skeletal muscle and time points included in the in vivo injury time course (III). (B) Dot plots showing analysis for (II) GFP expression in CD45 − /CD11b − /Ter119 − /CD31 − /Sca1 − /β1-Integrin + /CXCR4 + fresh SCs (I) from uninjured muscles of wild-type (FMO-WT, top) or Fos GFP (bottom) mice. SSC-A, side scatter area. Data were pre-gated on physical and live cell parameters (see for details). (C) Mean (±SD) percentage of fresh Fos GFP SCs expressing GFP (compiled analysis from 15 mice). (D) Pre-fixed (bottom) or non-pre-fixed (standard isolation, top) single fibers co-stained for PAX7 (green), FOS (red), and DAPI (blue). (E) Quantification (mean ± SD) of the percentage of PAX7+ SCs expressing FOS in freshly isolated single fibers stained as in (D). Data represent enumeration of more than 100 SCs across a minimum of 30 fibers per biological replicate for each condition (n = 3 mice per condition). (F) Fresh-frozen muscle sections co-stained for PAX7 (green), FOS (red), and DAPI (blue) 0, 1.5, and 12 h after cardiotoxin (CTX; 10 μM) injury. All channels are shown separately for the 1.5 h post-injury field (bottom row) and merged for 0, 1.5, and 12 h (top row). (G and H) Quantification of immunofluorescence (IF) data shown in (F), including (G) mean (±SD) percentage of PAX7+ SCs expressing FOS protein and (H) mean (±SD) number of PAX7+ SCs quantified per TA/ extensor digitorum longus (EDL) section per condition (n = 3 mice per time point). Student’s two-tailed unpaired t test (E) and one-way ANOVA with Tukey post hoc test (G and H). The scale bars represent 50 mm (D) and 100 μm (F). See also .

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: In Vivo, Expressing, Muscles, Isolation, Staining, Immunofluorescence, Two Tailed Test

Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: (A) Schematic showing the experimental design and FACS gating strategy for isolation of 1,000 Fresh Fos GFP+ and 1,000 Fos GFP− SCs directly sorted for RNA-seq analysis (SCs isolated from 4 mice). (B) Hierarchically clustered heatmap showing all 3,387 differentially expressed genes (DEGs; >1.5 FC, FDR < 0.05) in Fos GFP+ versus Fos GFP− SCs. (C) Volcano plot highlighting known SC regulator genes enriched (blue) or depleted (red) in fresh Fos GFP+ SCs. Notable mRNAs not significantly changed are indicated in black. (D) Top ranked Biocarta pathways associated with enriched genes in Fos GFP+ fresh SCs. (E) Venn diagrams showing overlap in genes enriched in Fos GFP+ or Fos GFP− SCs and in T3 (standard isolation, top) or T0 ( in-situ-fixed , quiescent SCs, bottom) SCs, respectively . The p values were determined by Fisher’s exact test of significance. (F) Heatmap of MAPK targets expressed in Fos GFP+ SCs relative to Fos GFP SCs. (G) Strategy for testing whether p38 MAPK induces FOS in freshly isolated single fibers. (H) Single fibers co-stained for PAX7 and FOS after isolation in the presence of vehicle or the p38 MAPK inhibitor SB202190 (SB). Scale bar, 50 μm. (I) Mean (±SD) percentage of PAX7+ SCs expressing FOS protein after isolation under the indicated condition. Data represent enumeration of more than 100 SCs across a minimum of 30 fibers per biological replicate for each condition (n = 3 mice per condition). The Z score equals the number of SDs from the mean expression of all genes (C and F). Fisher’s exact test (E) and Student’s two-tailed unpaired t test (I). See also .

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: Isolation, RNA Sequencing, In Situ, Staining, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: (A) 1,500 Fos cKO or 1,500 fresh control SCs were cultured for 7 days in GM. Shown is quantification of the mean (±SD) number of Hoechst+ cells per well (n = 3 mice per genotype). (B) 2,000 fresh Fos cKO or 2,000 control SCs cultured in GM for 4 or 7 days and pulsed with EdU 3 h before harvest, displaying EdU-positive (magenta) and Hoechst-positive (blue) nuclei and mean (±SD) percentage of EdU+ nuclei among total Hoechst+ cells after 4 (n = 2–4 mice per genotype) or 7 days (n = 3 mice per genotype) in culture. (C) Schematic showing the tamoxifen (TAM) treatment regimen before and after a freeze muscle injury (cryoinjury) in the TA muscle. (D) Representative Laminin-stained Fos cKO and control muscle sections (20×) from uninjured mice (top) and from mice 7 (center) and 50 (bottom) days after freeze injury. (E and F) Distribution (E) and mean (±SD) cross-sectional area (CSA; F) of fiber sizes from uninjured Fos cKO or control animals (n = 4 mice per genotype). (G and H) Distribution (G) and mean (±SD) CSA (H) of regenerating (centrally nucleated) muscle fibers 7 days after freeze injury (n = 5 mice per genotype). (I) Quantification of the total number of muscle fibers per TA/EDL section in control and Fos cKO mice before (left) and 50 days after freeze injury (right) (n = 5 mice per genotype per condition). (J) Total number of Pax7+ SCs in uninjured and injured (50 dpi, freeze) TA/EDL muscle sections of control (left) and Fos cKO mice (right) (n = 5 mice per genotype per condition). Dots represent data for individual control or Fos cKO animals overlaid with mean ± SD. Student’s two-tailed unpaired (A, B, F, H, and I) and paired (J) t test and Mann-Whitney U test (E and G). Scale bars, 100 μm (A, B, and D). See also .

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: Control, Cell Culture, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: (A) RNA-seq (normalized read counts) showing mean (±SD) Art1 mRNA expression in fresh Fos cKO and control SCs. (B) ChIP-qPCR assays using a FOS or immunoglobulin G (IgG)-only antibody to immunoprecipitate chromatin isolated from cultured SCs ectopically expressing FOS(+FOS) or GFP (+GFP) (n = 3 independent ChIP experiments using SCs from 3 mice). 5 different probes targeted the Art1 promoter near the FOS/AP-1 DNA motif. (C) RNA-Seq (normalized read counts) showing mean (±SD) Art1 mRNA expression in fresh SCs isolated from in-situ -fixed (T0) or non-pre-fixed (standard, T3) skeletal muscle . (D) qPCR showing mean (±SD) Art1 mRNA expression (normalized to GAPDH) in fresh relative to 7-day-cultured SCs. (E) 3,000 fresh SCs were cultured for 3 days with vehicle or MIBG (50 μM). Shown are images and quantification of the mean (±SD) percentage of EdU+ nuclei among Hoechst+ SCs (top). 3,000 fresh SCs were cultured for 6 days with vehicle or MIBG (50 μM). Shown are images and quantification of the total number (mean ± SD) of Hoechst+ nuclei (bottom) (n = 4 mice each condition). (F) 3,000 fresh SCs were infected with lentivirus on the day of isolation with a non-targeting control (NTC) shRNA or one of two distinct shRNAs targeting Art1 mRNA and cultured for 6 days. Shown are images and quantification of the mean percentage (±SD) of EdU+ nuclei among Hoechst+ nuclei (top) or the total number (mean ± SD) of Hoechst+ nuclei (bottom) (n = 4 mice per condition). (G) Distribution of ADP ribosylation levels (corrected total cell fluorescent signal [CTCF]) on individual vehicle/MIBG-treated (left), control/Fos cKO (center), and shNTC/sh Art1 -expressing (right) SCs cultured for 3 days. IF was performed under non-permeabilization conditions to ensure extracellular signal. n = 250 cells (vehicle)/80 cells (MIBG) from 3 mice, n = 588 cells (control)/395 cells (Fos cKO ) from 4 mice, and n = 1,300 cells (shNTC)/695 cells ( shArt1 ) from 4 mice. Red lines represent the median value, and lower and upper black lines represent the first and third quartiles of the data. shArt1 -expressing SCs have reduced cell-surface ADP ribosylation in a small subset of the population, specifically in the first quartile (highlighted in ). Shown are three representative images of cellsurface ADP ribosylation (green) on Hoechst+ (blue) control and Fos cKO fresh SCs after 3 days in culture. (H) Experimental design. We performed two consecutive injections for 2 days (1 injection per day) of MIBG (50 μM) or vehicle into the TA muscle following a CTX injury and then harvested regenerating muscle 7 dpi. (I) Representative images showing PAX7+ SCs associated with regenerating (centrally nucleated) fibers 7 days after CTX injury. Pax7 (red), nuclei (DAPI), and Laminin (Green) are shown. (J) Quantification of the total number of PAX7+ SCs per TA/EDL muscle section in vehicle- and MIBG-treated mice (n = 5 mice per treatment). (K and L) Enumeration of the mean CSA (K) and distribution (L) of regenerating (centrally nucleated) muscle fibers in vehicle- and MIBG-treated mice at 7 dpi (n = 5 mice per condition). Two-way ANOVA with post hoc Holm-Sidak test (B), one-way ANOVA with Tukey post hoc test (F), Student’s two-tailed unpaired t test (D, E, J, and K), and Mann-Whitney U test (G and L). Scale bars represent 100 μm (E and F), 10 mm (G), and 50 μm (I). See also .

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: RNA Sequencing, Expressing, Control, ChIP-qPCR, Isolation, Cell Culture, In Situ, Infection, shRNA, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: FOS licenses early events in stem cell activation driving skeletal muscle regeneration

doi: 10.1016/j.celrep.2020.108656

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-mouse PAX7 , DSHB , RRID: AB_528428.

Techniques: Control, Recombinant, Lysis, Protease Inhibitor, DNA Library Preparation, cDNA Synthesis, SYBR Green Assay, Imaging, Transfection, Virus, Plasmid Preparation, Immunodetection, Magnetic Beads, Microarray, Cloning, Software

Journal: PLoS ONE

Article Title: A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation

doi: 10.1371/journal.pone.0216167

Figure Lengend Snippet: (A ) Relative expression (mean ± SEM of Fragments Per Kilobase of transcript per Million mapped reads [FPKM]) of Rgs12 , Rgs14 , and Pax7 gene transcripts within indicated ages of mouse gastrocnemius muscle samples was obtained via RNA-Seq using an Illumina HiSeq 2000. Based on Gene Expression Omnibus dataset GSE108402 . Inset , detection of RGS12 protein expression via immunoblotting of ( a ) 100 μg of whole gastrocnemius muscle lysate from a 3-month-old C57BL/6J mouse and ( b ) 50 μg of lysate from myoblasts isolated from cardiotoxin-injected tibialis anterior (TA) muscle. (B) Normalized expression levels (mean ± SEM) of Rgs12 , Rgs14 , and Pax7 gene transcripts within indicated ages of flow-sorted, Pax7 + mouse skeletal muscle satellite cells were obtained via an Affymetrix Mouse Gene 1.0 ST microarray, based on Gene Expression Omnibus dataset GSE47401; published in . (C) Relative expression of Rgs12 and eMHC in TA muscle following CTX-induced muscle damage. RNA was extracted from muscle at the indicated time points and quantified using qRT-PCR, with Gapdh abundance as an internal control. *, p < 0.01 Rgs12 level compared to time zero (one-way ANOVA with Dunnett’s test); #, p < 0.0001 eMHC level compared to time zero (one-way ANOVA with Dunnett’s test). Inset , TA muscle was injected with 0.1 ml of 10 μM cardiotoxin (CTX) diluted in PBS; contralateral, PBS-injected TA muscle was used as a control. After four days, the muscles were harvested and used for RGS12 protein expression analysis by immunoblotting (with GAPDH protein levels interrogated in parallel as a loading control). (D) C2C12 cell line cultures (4 x 10 5 cells/well) were maintained in growth medium (DMEM containing 10% fetal bovine serum [FBS]) for two days. Differentiation was induced by replacing growth medium with differentiation medium (DMEM containing 2% horse serum [HS] instead of FBS). Total RNA and protein lysates were separately isolated from cell cultures at the indicated time points (hours) after the switch to differentiation medium. Rgs12 mRNA and RGS12 protein levels were determined by qRT-PCR and immunoblotting, respectively. GAPDH mRNA and protein levels were used as internal controls for each experiment. **, p < 0.01; ***, p < 0.001 versus level observed at time zero (one-way ANOVA with Dunnett’s test).

Article Snippet: Two cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA): the C2C12 adherent myoblastic cell line (ATCC CRL-1772) derived from a C3H mouse; and, the RD adherent cell line (ATCC CCL-136) derived from a rhabdomyosarcoma of a 7-year-old female Caucasian and exhibiting an unstable karyotype ( i . e ., hyperdiploid with a bimodal stemline number of 49 and 50 chromosomes).

Techniques: Expressing, RNA Sequencing, Gene Expression, Western Blot, Isolation, Injection, Microarray, Quantitative RT-PCR, Control, Muscles

Journal: PLoS ONE

Article Title: A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation

doi: 10.1371/journal.pone.0216167

Figure Lengend Snippet: (A) Immunoblotting (IB) of cell lysates from all three cell types indicates RGS12 protein expression, as detected using a rabbit anti-RGS12 polyclonal antibody previously described ; β-tubulin protein levels were also examined by immunoblotting in parallel as a loading control. (B) Cultures of the poorly differentiating, human rhabdomysarcoma RD cell line and the more-easily differentiated, mouse C2C12 cell line were separately fixed and stained with DAPI; RGS12 protein was detected by indirect immunofluorescence using UNC60-26.2.1. Panels (i-vi) represent: (i, iv) anti-RGS12 antibody detection with Alexa-fluor-594 secondary antibody; (ii, v) DAPI nuclear stain; and, (iii, vi) overlay of both images. (C) Endogenous RGS12 localizes to early (APPL1-positive) endosomes within C2C12 cells. C2C12 cell line cultures (4 x 10 5 cells/well) were maintained in growth medium (DMEM containing 10% fetal bovine serum [FBS]) for two days. Cells were then fixed in paraformaldehyde, permeabilized, and stained with primary mouse monoclonal antibody UNC60-26.2.1 and secondary Alexa-fluor-594 anti-mouse antibody, alone or in combination with primary anti-APPL1 or -Rab9 rabbit polyclonal antibodies followed by Alexa-fluor-488 secondary anti-rabbit antibody. Overlays in panels (vii-ix) are absent the DAPI nuclear stain ( blue ) to highlight lack of overlap between RGS12 and Rab9 signals. (D) RGS12 N-terminus binds to select phosphatidylinositides in a lipid dot-blot protein overlay assay. A “PIP Strip” nitrocellulose membrane pre-spotted with the indicated phospholipid species was probed with 20 μg/mL of GST alone, recombinant GST-RGS12 protein (amino-acids 9–406 spanning PDZ and PTB domains; “WT”), or GST-RGS12(aa 9–406) protein with alanine substitutions to four arginines in the PTB domain (Arg-255, -260, -262, and -308; “4R→A”) previously predicted by electrostatic contouring to be involved in phospholipid binding. After extensive washing, the binding of protein to phospholipid spots was detected by chemiluminescence using anti-GST mouse monoclonal antibody and anti-mouse-horseradish peroxidase conjugated secondary antibody. Lipid abbreviations: LPA, lysophosphatidic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.

Article Snippet: Two cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA): the C2C12 adherent myoblastic cell line (ATCC CRL-1772) derived from a C3H mouse; and, the RD adherent cell line (ATCC CCL-136) derived from a rhabdomyosarcoma of a 7-year-old female Caucasian and exhibiting an unstable karyotype ( i . e ., hyperdiploid with a bimodal stemline number of 49 and 50 chromosomes).

Techniques: Western Blot, Expressing, Control, Staining, Immunofluorescence, Dot Blot, Overlay Assay, Stripping Membranes, Membrane, Recombinant, Binding Assay

Journal: PLoS ONE

Article Title: A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation

doi: 10.1371/journal.pone.0216167

Figure Lengend Snippet: (A) Immunoblotting (IB) of cell lysates from three stable clones of the C2C12 cell line indicating their stable expression of a constitutively-active, GTPase-deficient (G12V) mutant of H-Ras protein (via detection of its N-terminal myc-epitope tag). (B) Co-immunoprecipitation of myc-tagged, activated H-Ras (G12V mutant) with endogenous RGS12 protein expressed in the C2HRas cell line clone 9. IP: immunoprecipitation; IB: immunoblotting. (C) To test the effect of stable expression of activated H-Ras on myoblast differentiation in vitro , multi-nucleated myotube content of indicated cell populations (either parental C2C12 cells [panels i, ii] or C2HRas clone #9 cells [panels iii, iv]) was measured by fixation and staining for sarcomere myosin (MHC; green ) and nuclear DNA content (DAPI; pseudocolored red ), either pre-differentiation (panels i, iii) or post-differentiation by culture for 5 days in low serum (2% horse serum; panels ii, iv). The mean fusion index for several C2C12 cell populations was 40% (± 2.5%; SEM), consistent with other reports [ , ]. In contrast, the C2HRas cell line clone 9 was incapable of myotube formation (panel iv) and no fusion index could be calculated. (D) Co-immunoprecipitation of myc-tagged, activated H-Ras (G12V mutant) with ectopically co-expressed, HA-tagged RGS12 in C2C12 cells. IgH: immunoglobulin heavy-chain. (E) Endogenous levels of RGS12 protein are down-regulated during C2C12 differentiation into myotubes ( i . e ., 7 days of culture in low serum medium), whereas the same culture conditions did not lower RGS12 levels within the C2HRas cell line clone #9. (F) Mammalian RGS12 proteins encode a “destruction box” recognition motif, conforming to the consensus RxxLxxxx(D/N), where “x” is any amino-acid, that is found in most substrates of the Anaphase-Promoting Complex (APC). This destruction box sequence is completely conserved across human (h), mouse (m), and rat (r) RGS12 proteins (i.e., amino acids 402–410 of mouse RGS12: R AF L DGDA D ); this consensus motif is preserved in zebrafish (z) and Drosophila (d) RGS12 orthologs, as well as in the APC targets Skp2 and Myf5.

Article Snippet: Two cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA): the C2C12 adherent myoblastic cell line (ATCC CRL-1772) derived from a C3H mouse; and, the RD adherent cell line (ATCC CCL-136) derived from a rhabdomyosarcoma of a 7-year-old female Caucasian and exhibiting an unstable karyotype ( i . e ., hyperdiploid with a bimodal stemline number of 49 and 50 chromosomes).

Techniques: Western Blot, Clone Assay, Expressing, Mutagenesis, Immunoprecipitation, In Vitro, Staining, Sequencing

Journal: PLoS ONE

Article Title: A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation

doi: 10.1371/journal.pone.0216167

Figure Lengend Snippet: (A) RGS12-overexpressing and control C2C12 cell line cultures were each switched from growth medium (DMEM, 10% FBS) to differentiation medium (DMEM, 2% horse serum) and cultured for two days. Cells were then fixed with paraformaldehyde and stained with anti-MHC antibody (MF20) and Alexa-fluor-594 secondary antibody ( red ). Nuclei were visualized with DAPI staining ( blue ). (B) Differentiation to myotubes was quantitated by the fusion index [36, 42): namely, the percentage of nuclei present in fused, MHC-positive cells vs total nuclei in the field. Graphed below the fusion index is the quantitation of MHC-positive cells observed in each field examined. Statistical significance was tested by one-way ANOVA: **, p<0.01; ***, p<0.001. (C) Expression of epitope-tagged, full-length RGS12 was confirmed by immunoblotting (IB) with indicated anti-epitope tag antibodies. (D) Ectopic expression of Myc-epitope tagged, full-length RGS12 was verified by immunoblotting (IB) of whole cell lysates; equal total protein loading was verified by β-tubulin detection. (E) RGS12-overexpressing and mock transfected (empty vector) C2C12 cell line cultures in growth medium (DMEM, 10% FBS) were separately incubated for 60 minutes with 5-ethynyl-2’-deoxyuridine (EdU) to detect DNA synthesis by proliferating cells. Click-iT labeling with Alexa-fluor-488 identified cells with newly synthesized DNA. The percentage of EdU positive cells were counted using ImageJ software. N = 3 independent experiments. Statistical significance was established by Student’s t-test: ***, p<0.001. (F) C2C12 cell cultures were infected with lentiviridae encoding either a non-specific (NS) control shRNA or one of two different shRNAs targeting Rgs12 (#2, #5), and then selected in puromycin-containing growth medium (DMEM + 10% FBS) for two weeks. Cell cultures were then switched from growth medium to differentiation medium (DMEM + 2% HS) for five days; as a positive control for low-to-nil fusion index, the poorly-differentiating, human RD cell line was also cultured for five days in differentiation medium (rightmost panel). Cell cultures were immunolabeled with a primary antibody directed against sarcomere MHC (MF20) and Alexa-fluor-594 secondary antibody; nuclei were counterstained with DAPI. ( G ) Underneath each fluorescence micrograph is the plot of phospho-ERK content (quantified by densitometry and normalized to total ERK content) from parallel cultures harvested at indicated timepoints; immunoblot inset within graph presents representative data from nontransfected RD cells over five days of culture. (H) shRNA-mediated knockdown of RGS12 expression was confirmed by immunoblotting of whole cell lysates from indicated, shRNA-expressing C2C12 cell lines; β-actin protein levels were also examined by immunoblotting in parallel as a loading control. (I) Myotube formation within indicated C2C12 cell cultures was quantitated by calculation of the fusion index: nuclei from sarcomere MHC-positive, multi-nucleated cells and MHC-negative, non-fused cells were separately counted using ImageJ software and the fusion index calculated as the ratio of nuclei present in fused MHC-positive cells to the total number of nuclei in the field (expressed as a percentage). ***, p < 0.005 versus control shRNA-expressing C2C12 cell line; Student’s t-test.

Article Snippet: Two cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA): the C2C12 adherent myoblastic cell line (ATCC CRL-1772) derived from a C3H mouse; and, the RD adherent cell line (ATCC CCL-136) derived from a rhabdomyosarcoma of a 7-year-old female Caucasian and exhibiting an unstable karyotype ( i . e ., hyperdiploid with a bimodal stemline number of 49 and 50 chromosomes).

Techniques: Control, Cell Culture, Staining, Quantitation Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Incubation, DNA Synthesis, Labeling, Synthesized, Software, Infection, shRNA, Positive Control, Immunolabeling, Fluorescence, Knockdown

Journal: iScience

Article Title: Dnmt3a overexpression disrupts skeletal muscle homeostasis, promotes an aging-like phenotype, and reduces metabolic elasticity

doi: 10.1016/j.isci.2025.112144

Figure Lengend Snippet: Dnmt3a regulates the Akt-FoxO-atrogene axis and causes loss of sensitivity to starvation (A) PPI network of differentially expressed genes (DEGs) with FDR <0.05 and fold-change value >2 upregulated in Dnmt3a-Tg muscle compared with WT muscle (1,534 nodes and 12,704 edges are shown). Node color indicates log 2 fold-change value, and circle size of nodes indicates number of direct edges (degree). (B) The top 20 putative hub genes among the network of DEGs upregulated in Dnmt3a-Tg muscle were identified by PPI network analysis. (C) Relative mRNA expression of Akt1 in gastrocnemius muscle from young (3-month-old) WT and age-matched Dnmt3a-Tg mice ( n = 8 mice/group). (D) GSEA of a gene set of muscle FoxO signaling and putative FoxO1 target in skeletal muscle from microarray data of young (3-month-old) WT and Dnmt3a-Tg muscles ( n = 4 mice/group). (E) Relative mRNA expression of FoxO signaling pathway and atrophy-related genes in gastrocnemius muscle from young (3-month-old) WT and Dnmt3a-Tg mice ( n = 8 mice/group). (F) Weights of skeletal muscles (gastrocnemius, quadriceps, TA, EDL, and soleus) from fed and 48-h-fasted WT and Dnmt3a-Tg female mice ( n = 10–11 fed and 48-h-fasted WT mice, n = 5 fed and 48-h-fasted Dnmt3a-Tg mice). (G) Starvation-induced difference in skeletal muscle mass of gastrocnemius and EDL muscles in WT and Dnmt3a-Tg female mice ( n = 11 WT mice, n = 5 Dnmt3a-Tg mice). (H–J) Representative immunoblot images (H) and densitometric analysis (I, J) of Dnmt3a, p-Akt (Ser473), AKT, p-FoxO1 (Ser256), FoxO1, C/EBPδ, Atrogin1, 4EBP1, LC3b, and ubiquitin protein in the gastrocnemius of fed and 48-h-fasted WT and Dnmt3a-Tg female mice ( n = 7 fed and 48-h-fasted WT mice, n = 5 fed and 48-h-fasted Dnmt3a-Tg mice). (K) Relative mRNA expression of atrogenes downstream of FoxO in gastrocnemius muscles from WT and Dnmt3a-Tg mice ( n = 10–11 fed and 48-h-fasted WT mice, n = 5 fed and 48-h-fasted Dnmt3a-Tg mice). All data indicate mean ± SE. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (C, E, G) Student’s two-tailed unpaired t test. (F, I–K) Two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. F, fed; S, starvation; WT, wild type; Tg: Dnmt3a-Tg.

Article Snippet: Rabbit anti-phospho-Akt (Ser473) , Cell Signaling , Cat# 9271; RRID: AB_329825.

Techniques: Expressing, Microarray, Muscles, Western Blot, Ubiquitin Proteomics, Two Tailed Test

Journal: iScience

Article Title: Dnmt3a overexpression disrupts skeletal muscle homeostasis, promotes an aging-like phenotype, and reduces metabolic elasticity

doi: 10.1016/j.isci.2025.112144

Figure Lengend Snippet:

Article Snippet: Rabbit anti-phospho-Akt (Ser473) , Cell Signaling , Cat# 9271; RRID: AB_329825.

Techniques: Virus, Recombinant, Injection, Immunodetection, Plasmid Preparation, Fluorescence, Lysis, Bicinchoninic Acid Protein Assay, Western Blot, Membrane, Sterility, Gene Expression, Microarray, Labeling, Hybridization, Ligation, Purification, DNA Labeling, Methylation, Library Quantification, DNA Ligation, Transfection, Methylation Sequencing, Software, Functional Assay, Mass Spectrometry, Real-time Polymerase Chain Reaction, Imaging, Microscopy

Journal: iScience

Article Title: A Tead1-Apelin axis directs paracrine communication from myogenic to endothelial cells in skeletal muscle

doi: 10.1016/j.isci.2022.104589

Figure Lengend Snippet: A transcription factor screen identifies Tead1 as a direct regulator of apelin transcription (A) Overview of the yeast one-hybrid (Y1H) assay to screen 745 transcription factors for their ability to interact with the Apelin ( Apln ) promoter. Binding of a transcription factor is readout via expression of the HIS3 reporter which enables yeast growth on a selective 3AT-containing medium plate. (B) Y1H screen results using the −200/-1 bp fragment of the mouse Apln promoter as bait and 745 mouse TFs as prey (n = 4 replicates per tested TF, hence the formation of a quadrant in case of positive interaction). (C) Z-score-normalized Y1H spot intensities for all 745 TFs. Six TFs with Z-scores above the background threshold (red line) are noted here and in (B). (D) Relative mRNA expression of the six TF candidates in bulk mRNA profiling of human and mouse tissues. Microarray data for humans (left) from the GeneAtlas UI33A and mouse (right) from the GeneAtlas MOE430 of the bioGPS gene annotation portal. n = 2 replicates per tissue. Expression data are normalized and presented in a log2-scaled heatmap by species. (E) mRNA expression of Apln and the six TFs in C2C12 myoblasts measured by RT-qPCR relative to Hprt . n.d., not detected. Mean ± SE of mean (SEM) of n = 16 replicates. (F) mRNA expression of Tead1 in C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs for 3 d n = 16 replicates. (G) ChIP-qPCR assay of Tead1 testing for binding to known target promoters ( Ctgf , Ankrd1 ), Apln promoter (−177/-77 bp), or a random negative control in C2C12 myoblasts treated with either scrambled control or Tead1 -targeted siRNA for 3 days. ChIP was performed with Tead1 or IgG control antibodies and qPCR was normalized to IP input. n = 1 biological replicate. (H) Luciferase activity of five Apln promoter fragments transfected into C2C12 myoblasts at D3 with scrambled or Tead1 siRNA. Vector control contains no Apln promoter. Mean ± SEM of n = 8 biological replicates. p values are reported from two-tailed, unpaired t-tests between siRNA conditions.

Article Snippet: Apln taqman probe , Life Technologies , Mm00443562_m1.

Techniques: Y1H Assay, Binding Assay, Expressing, Microarray, Quantitative RT-PCR, Transfection, Control, ChIP-qPCR, Negative Control, Luciferase, Activity Assay, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: A Tead1-Apelin axis directs paracrine communication from myogenic to endothelial cells in skeletal muscle

doi: 10.1016/j.isci.2022.104589

Figure Lengend Snippet: Apelin is repressed by Tead1 in muscle cells in vitro and in vivo (A) Immunostaining of Apln protein during C2C12 myotube differentiation. Scale bars, 100 μm. (B) Quantification of Apln mRNA by RT-qPCR during C2C12 myotube differentiation relative to Hprt using 2 –dCt method. n = 4 cell culture replicates per time point. (C and D) Quantification of apelin mRNA by RT-qPCR (C) or apelin peptide in supernatant by ELISA (D) in C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs for 3days n = 16-20 replicates per condition. (E–I) Analysis of Tead1 and in adult mice over-expression Tead1 in skeletal muscle myofibers under the muscle creatine kinase (MCK) promoter (MCK-OE-Tead1 mice), compared to WT C57BL6 controls. (F and G) Tead1 mRNA (F) and Apln mRNA (G) expression levels were measured by RT-qPCR and normalized to Hprt in tibialis anterior (TA) muscles. n = 6 mice per condition. (H and I) Apln peptide concentration measured by ELISA in TA muscles (H) or serum (I). n = 5 mice per condition for TA; n = 11 mice per condition for serum. All data are presented as mean ± SEM, and p values are reported from two-tailed, unpaired t-tests between conditions. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Apln taqman probe , Life Technologies , Mm00443562_m1.

Techniques: In Vitro, In Vivo, Immunostaining, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Control, Over Expression, Expressing, Muscles, Concentration Assay, Two Tailed Test

Journal: iScience

Article Title: A Tead1-Apelin axis directs paracrine communication from myogenic to endothelial cells in skeletal muscle

doi: 10.1016/j.isci.2022.104589

Figure Lengend Snippet: Apln, Aplnr, and Tead1 expression dynamics in regenerating skeletal muscle (A and B) Single-cell RNA-sequencing analysis of a notexin injury response in TA muscle adult mice. TA muscle samples from 0, 2, 5, and 7 days post-injury (d.p.i.) with n = 2-3 mice per time-point were analyzed from De Micheli et al. (2020) (A) UMAP projection of scRNAseq data demonstrating cell-type annotations of clusters using markers shown in Figure S3 . (B) Dot plots showing expression of Apln , Aplnr , and Tead1 by cell-type cluster and time-point. Dot size shows the frequency of cells expressing non-zero transcript level. Dot color shows average expression level. (C and D) In vitro expression of Apln and Aplnr protein by immunofluorescence (C) and mRNA by qRT-PCR (D) in C166 endothelial cells (ECs) and C2C12 myotubes differentiated for 8days n = 5 for C166 ECs; n = 4 for C2C12 myotubes. Scale bar, 100 μm. (E–I) Regeneration of TA muscles of adult WT mice after IM injection of glycerol analyzed at 0, 3, 7, and 14 days.p.i. by gene expression microarray and immunohistology. (E) Experimental overview. (F and G) Apln and Aplnr mRNA levels from transcriptomic analyses, normalized and presented as fold-change relative to mean of 0 days.p.i. Data are mean ± SEM of n = 5 (0, 14 days.p.i.) and n = 6 (3, 7 days.p.i.) mice. (H) Representative images of CD31 and Laminin immunostaining in regenerating muscle samples at 0, 3, 7, and 14 days.p.i. Scale bar, 50 μm. (I) Quantification of CD31 + endothelial cells per cross-sectional area. Data are mean ± SEM of n = 5 TA muscles. In (F–G) and (I), p values are reported by two-tailed unpaired t-test compared to 0 days.p.i.; with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Apln taqman probe , Life Technologies , Mm00443562_m1.

Techniques: Expressing, RNA Sequencing, In Vitro, Immunofluorescence, Quantitative RT-PCR, Muscles, Injection, Gene Expression, Microarray, Immunostaining, Two Tailed Test

Journal: iScience

Article Title: A Tead1-Apelin axis directs paracrine communication from myogenic to endothelial cells in skeletal muscle

doi: 10.1016/j.isci.2022.104589

Figure Lengend Snippet: Apln promotes endothelial cell remodeling in vitro and in vivo (A and B) EC expansion of C166 and EOMA lines treated for 5days with the recombinant Apln-13 peptide at 10μM. (A) Representative images of DAPI staining of C166 ECs. Scale bar, 100 μm. (B) Quantification of the total number of C166 and EOMA cells treated with Apln compared to the control medium. (n = 16 cell culture replicates per group). (C–F) Daily Apln-13 administration at 0.5 μmol/kg/day for 7days in aged mice following cardiotoxin-induced injury in TA muscle. (D) Expression of Pecam1 mRNA by RT-qPCR in whole TA muscles at 3 and 7 days.p.i. and non-injured TA muscles (n = six to seven TA muscles per group). (E) Representative images of CD31 and Laminin immunostaining in CTX-injured TA muscles at 3 days.p.i. Scale bar, 50 μm. (F) Quantification of CD31 + endothelial cells at 3 and 7 days.p.i. in regenerating regions (n = 5 TA muscles per group). Dashed line indicates the basal level of CD31 + cell per mm 2 in non-injured TA muscles (from Figure 3 I). In (B), (D) and (I), p values are reported by two-tailed unpaired t-test compared to 0 days.p.i.; n.s represents p > 0.05.

Article Snippet: Apln taqman probe , Life Technologies , Mm00443562_m1.

Techniques: In Vitro, In Vivo, Recombinant, Staining, Control, Cell Culture, Expressing, Quantitative RT-PCR, Muscles, Immunostaining, Two Tailed Test

Journal: iScience

Article Title: A Tead1-Apelin axis directs paracrine communication from myogenic to endothelial cells in skeletal muscle

doi: 10.1016/j.isci.2022.104589

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Article Snippet: Apln taqman probe , Life Technologies , Mm00443562_m1.

Techniques: Luciferase, Multiplex Assay, DNA Purification, Recombinant, Software

Journal: iScience

Article Title: Dnmt3a overexpression disrupts skeletal muscle homeostasis, promotes an aging-like phenotype, and reduces metabolic elasticity

doi: 10.1016/j.isci.2025.112144

Figure Lengend Snippet: Dnmt3a regulates the Akt-FoxO-atrogene axis and causes loss of sensitivity to starvation (A) PPI network of differentially expressed genes (DEGs) with FDR <0.05 and fold-change value >2 upregulated in Dnmt3a-Tg muscle compared with WT muscle (1,534 nodes and 12,704 edges are shown). Node color indicates log 2 fold-change value, and circle size of nodes indicates number of direct edges (degree). (B) The top 20 putative hub genes among the network of DEGs upregulated in Dnmt3a-Tg muscle were identified by PPI network analysis. (C) Relative mRNA expression of Akt1 in gastrocnemius muscle from young (3-month-old) WT and age-matched Dnmt3a-Tg mice ( n = 8 mice/group). (D) GSEA of a gene set of muscle FoxO signaling and putative FoxO1 target in skeletal muscle from microarray data of young (3-month-old) WT and Dnmt3a-Tg muscles ( n = 4 mice/group). (E) Relative mRNA expression of FoxO signaling pathway and atrophy-related genes in gastrocnemius muscle from young (3-month-old) WT and Dnmt3a-Tg mice ( n = 8 mice/group). (F) Weights of skeletal muscles (gastrocnemius, quadriceps, TA, EDL, and soleus) from fed and 48-h-fasted WT and Dnmt3a-Tg female mice ( n = 10–11 fed and 48-h-fasted WT mice, n = 5 fed and 48-h-fasted Dnmt3a-Tg mice). (G) Starvation-induced difference in skeletal muscle mass of gastrocnemius and EDL muscles in WT and Dnmt3a-Tg female mice ( n = 11 WT mice, n = 5 Dnmt3a-Tg mice). (H–J) Representative immunoblot images (H) and densitometric analysis (I, J) of Dnmt3a, p-Akt (Ser473), AKT, p-FoxO1 (Ser256), FoxO1, C/EBPδ, Atrogin1, 4EBP1, LC3b, and ubiquitin protein in the gastrocnemius of fed and 48-h-fasted WT and Dnmt3a-Tg female mice ( n = 7 fed and 48-h-fasted WT mice, n = 5 fed and 48-h-fasted Dnmt3a-Tg mice). (K) Relative mRNA expression of atrogenes downstream of FoxO in gastrocnemius muscles from WT and Dnmt3a-Tg mice ( n = 10–11 fed and 48-h-fasted WT mice, n = 5 fed and 48-h-fasted Dnmt3a-Tg mice). All data indicate mean ± SE. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (C, E, G) Student’s two-tailed unpaired t test. (F, I–K) Two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. F, fed; S, starvation; WT, wild type; Tg: Dnmt3a-Tg.

Article Snippet: Rabbit anti-phospho-FoxO1 (Ser256) , Cell Signaling , Cat# 9461; RRID: AB_329831.

Techniques: Expressing, Microarray, Muscles, Western Blot, Ubiquitin Proteomics, Two Tailed Test

Journal: iScience

Article Title: Dnmt3a overexpression disrupts skeletal muscle homeostasis, promotes an aging-like phenotype, and reduces metabolic elasticity

doi: 10.1016/j.isci.2025.112144

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Article Snippet: Rabbit anti-phospho-FoxO1 (Ser256) , Cell Signaling , Cat# 9461; RRID: AB_329831.

Techniques: Virus, Recombinant, Injection, Immunodetection, Plasmid Preparation, Fluorescence, Lysis, Bicinchoninic Acid Protein Assay, Western Blot, Membrane, Sterility, Gene Expression, Microarray, Labeling, Hybridization, Ligation, Purification, DNA Labeling, Methylation, Library Quantification, DNA Ligation, Transfection, Methylation Sequencing, Software, Functional Assay, Mass Spectrometry, Real-time Polymerase Chain Reaction, Imaging, Microscopy

Journal: Scientific Reports

Article Title: Investigating the effects of chronic perinatal alcohol and combined nicotine and alcohol exposure on dopaminergic and non-dopaminergic neurons in the VTA

doi: 10.1038/s41598-021-88221-8

Figure Lengend Snippet: Topmost DEGs and their predicted miRNA targets following microarray expression analysis.

Article Snippet: Lypla2 (Assay ID: Rn00580197_m1), Bnip3l (Assay ID: Rn01534668_g1), Gnai2 (Assay ID: Rn01447850_m1), Gtf2i (Assay ID: Rn01499727_m1), Ndrg2 (Assay ID: Rn01414698_m1), Atp2a2 (Assay ID: Rn00568762_m1), Icmt (Assay ID: Rn01516590_m1), Ank1 (Assay ID: Rn01756750_m1), and Gramd1c (Assay ID: Rn01475288_m1).

Techniques: Microarray, Expressing, Binding Assay, Derivative Assay