Journal: PLoS ONE

Article Title: Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

doi: 10.1371/journal.pone.0114365

Figure Lengend Snippet: (A) Representative fluorescence images show 1 kb MYH4 promoter-reporter constructs are differentiation specific. (B) Equivalent 5′ deletion analysis of the pig and human MYH4 promoters. Promoter activities in Day 6 differentiated C2C12 myotubes (mean ± SD). * Indicates Human MYH4 promoter activity was significantly different to the equivalent length pig promoter ( p <0.05). (C) 1 kb pig and human MYH4 promoter activity in response to (mouse) MyoD over expression in day 5 differentiated C2C12 myotubes (mean ± SD). * Indicates MYH4 promoter activity was significantly different to the control transfection ( p <0.05).

Article Snippet: Protein-DNA binding conditions consisted of 50 ng/ul Poly dI-dC, 5% glycerol, 0.05% NP-40, 50 mM KCL, 1 mM MgCl 2 , 1 mM EDTA, 20 fmol biotin-labeled probe, 4 pmol un-labeled competitor probe and 3 μl of C2C12 myotube nuclear extract (from NE-PER extraction kit, Thermo Scientific) in 1x binding buffer (LightShift Chemiluminescent EMSA kit, Thermo Scientific) in a total volume of 20 μl.

Techniques: Fluorescence, Construct, Activity Assay, Over Expression, Control, Transfection

Journal: PLoS ONE

Article Title: Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

doi: 10.1371/journal.pone.0114365

Figure Lengend Snippet: (A) Site directed mutagenesis within the AT-rich region and the CArG-box of the 1 kb human MYH4 promoter (substitutions indicated by a “bolt” symbol). (B) Site directed mutagenesis within the CArG/Ebox2 region of the 1 kb human MYH4 promoter. A “bolt” indicates a single base pair substitution and a “cross” indicates removal of a single base pair. Promoter activities were measured in day 6 differentiated C2C12 myotubes (mean ± SD). Differing letters (a,b,c,d,e) constitute a significant difference between promoter activities ( p <0.05). Open bar = pig promoter; black bar = human promoter; grey bar = mutant human promoter.

Article Snippet: Protein-DNA binding conditions consisted of 50 ng/ul Poly dI-dC, 5% glycerol, 0.05% NP-40, 50 mM KCL, 1 mM MgCl 2 , 1 mM EDTA, 20 fmol biotin-labeled probe, 4 pmol un-labeled competitor probe and 3 μl of C2C12 myotube nuclear extract (from NE-PER extraction kit, Thermo Scientific) in 1x binding buffer (LightShift Chemiluminescent EMSA kit, Thermo Scientific) in a total volume of 20 μl.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

doi: 10.1371/journal.pone.0114365

Figure Lengend Snippet: (A) Electrophoretic mobility shift assay using a 62 bp biotin labeled probe (spanning the pig CArG and Ebox2 region; −91 bp to −31 bp relative to the TATA-box). Probes were incubated with C2C12 myotube nuclear extracts. Cross-species competition for bound proteins was conducted using shorter un-labeled pig and human probes. The human CArG-box is unable to bind proteins forming complex B with the pig CArG-box. Experiments were repeated 3 times to confirm the results. (B) Removal of the CArG-box region (total of 22 bp removed) from the 1 kb pig and human MYH4 promoters. Promoter activities were measured in day 6 differentiated C2C12 myotubes (mean ± SD). Differing letters (a,b,c) constitute a significant difference between promoter activities ( p <0.05).

Article Snippet: Protein-DNA binding conditions consisted of 50 ng/ul Poly dI-dC, 5% glycerol, 0.05% NP-40, 50 mM KCL, 1 mM MgCl 2 , 1 mM EDTA, 20 fmol biotin-labeled probe, 4 pmol un-labeled competitor probe and 3 μl of C2C12 myotube nuclear extract (from NE-PER extraction kit, Thermo Scientific) in 1x binding buffer (LightShift Chemiluminescent EMSA kit, Thermo Scientific) in a total volume of 20 μl.

Techniques: Electrophoretic Mobility Shift Assay, Labeling, Incubation

Journal: PLoS ONE

Article Title: Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

doi: 10.1371/journal.pone.0114365

Figure Lengend Snippet: (A) Alignment of the proximal pig and human MYH4 promoter. All base pair numbering is relative to the respective TATA-box (+1). Base pair mismatches, highlighted in black (with base pair numbering relative to the human TATA-box), were responsible for the differential activity of the 1 kb pig and human MYH4 promoter in C2C12 myotubes. Bold-underline indicates the 62 bp pig probe used in EMSA experiments (spanning the CArG-box and E-box region (−93 bp to −31 bp)). The two bold base pairs ( CC ) indicate the mutated HindIII site for generating chimeric-promoters. (B) Cross-species alignment of the CArG-box and E-box region from MYH4 expressing and non-expressing animals. Bases highlighted in the black columns indicate the location of crucial base pair differences required for high MYH4 expression in pigs relative to humans. There is no common mutation amongst MYH4 expressing and non-expressing species, but there are a number of differences (highlighted in grey) within this highly conserved region, which may be critical to the strength of promoter activation.

Article Snippet: Protein-DNA binding conditions consisted of 50 ng/ul Poly dI-dC, 5% glycerol, 0.05% NP-40, 50 mM KCL, 1 mM MgCl 2 , 1 mM EDTA, 20 fmol biotin-labeled probe, 4 pmol un-labeled competitor probe and 3 μl of C2C12 myotube nuclear extract (from NE-PER extraction kit, Thermo Scientific) in 1x binding buffer (LightShift Chemiluminescent EMSA kit, Thermo Scientific) in a total volume of 20 μl.

Techniques: Activity Assay, Expressing, Mutagenesis, Activation Assay

Journal: eLife

Article Title: The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( A ) Schematic overview of the strategy used to generate myotube templates with an associated timeline for downstream culture (made with BioRender). ( B ) Representative confocal stitched images of myotube templates labelled for sarcomeric α-actinin (SAA; magenta) at days 2, 5, 10, 14, 16, and 18 of culture. Scale bar, 1 mm. ( C ) Representative confocal image of myotubes at day 5 labelled with DAPI (cyan) and SAA (magenta). Scale bar, 50 µm. ( D ) Quantification of SAA area coverage (left axis; black line) and nuclear fusion index (right axis; grey line) of myotube templates at days 2, 5, 10, 14, 16, and 18 of culture. n=9–16 across N=3–6 independent biological replicates. Graph displays mean ± s.e.m.; one-way ANOVA with Tukey’s post-test, minimum ***p=0.002 (SAA coverage), ****p˂0.0001 (nuclear fusion index). ( E ) Optical density (OD) at 490 nm of media after myotube template incubation with MTS assay reagent on days 2, 5, 10, 14, 16, and 18 of culture. n=9–12 across N=3–4 independent biological replicates. Graph displays mean ± s.e.m.; one-way ANOVA with Tukey’s post-test, **p=0.0033. Raw data available in . Figure 1—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

Techniques: Incubation, MTS Assay

Journal: eLife

Article Title: The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( A ) Schematic overview of the engraftment of freshly isolated MuSCs and the timeline for downstream analysis (made with BioRender). ( B ) Representative confocal images of myotube templates (phalloidin: magenta) with engrafted MuSCs (YFP: yellow, Pax7: white, white arrows) at 1, 3, and 7 days post-engraftment (DPE). Scale bar, 50 µm. ( C ) Representative confocal image of a donor MuSC (DAPI: cyan, YFP: yellow, Pax7: white) indicated with a white arrow, and myotubes (phalloidin: magenta) at 7 DPE. Scale bar, 20 µm. ( D ) Quantification of mononuclear DAPI + YFP + Pax7 + cell density per mm 2 at 1, 3, and 7 DPE across different starting MuSC engraftment numbers (200, 500, 1500, and 2500). n=9–15 across N=3–5 independent biological replicates. Graph displays mean ± s.e.m.; one-way ANOVA with Dunnet’s test for each individual timepoint comparing against the 500 MuSC condition, **p=0.0025, 0.0051, 0.0029, ****p˂0.0001. Raw data available in . Figure 2—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

Techniques: Isolation

Journal: eLife

Article Title: The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( A ) Representative confocal image of a mononuclear cell (DAPI: cyan) positive for YFP (yellow), caveolin-1 (magenta), and c-FOS (white) at 1 day post-engraftment (DPE) (top), and a c-FOS - cell at 7 DPE (bottom). Scale bar, 20 µm. ( B ) Stacked bar graph showing proportions of c-FOS ± cells at 1, 3, and 7 DPE in the DAPI + YFP + Cav-1 + population. n=9 across N=3 independent biological replicates. Graph displays mean ± s.e.m. for c-FOS + and c-FOS - ; one-way ANOVA with Tukey’s post-test comparing the FOS - proportions of each timepoint, ****p˂0.0001. ( C ) Stacked bar graph showing proportions of Ki67 ± cells at 1, 3, and 7 DPE in the DAPI + YFP + Pax7 + population. n=10–11 across N=3–4 independent biological replicates. Graph displays mean ± s.e.m. for Ki67 + and Ki67 - ; one-way ANOVA with Tukey’s post-test comparing the Ki67 - proportions of each timepoint, ****p˂0.000.1. ( D ) Timeline of EdU/Ki67 co-labelling experiment (made with BioRender). ( E ) Stacked bar graph showing proportions of EdU ± cells at 7 DPE in the DAPI + YFP + Ki67 - mononuclear cell population. n=15 across N=5 independent biological replicates. Graph displays mean ± s.e.m. for EdU + and EdU - . ( F ) Representative confocal stitched images of myotube templates (sarcomeric α-actinin (SAA): magenta) 2 days after a 4 hr exposure to the physiological salt solution (PSS) control or a 2.4% barium chloride (BaCl 2 ) solution. Scale bar, 1 mm. ( G ) Proportion of Ki67 ± cells at 2 days post-injury (DPI) in the DAP + YFP + Pax7 + population. n=16, 18 across N=5, 6 biological replicates. Graph displays mean ± s.e.m. for Ki67 + and Ki67 - ; unpaired t-test of the Ki67 - proportions of both conditions, ****p˂0.0001. Raw data available in . Figure 3—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

Techniques:

Journal: eLife

Article Title: The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( A ) Key for figure icons. ( B–F ) Line graphs of mononucleated DAPI + YFP + Pax7 + cell density at 1, 3, and 7 days post-engraftment (DPE) (left) and pie charts showing the proportion of Ki67 ± cells at 7 DPE (right) for cells seeded into a two-dimensional (2D) microwell with a Geltrex coating ( B ), engrafted into 3D myotube templates on day 5 ( C ) vs. day 0 ( D ) of differentiation. Additional comparisons include engraftment into a 3D cellulose-reinforced extracellular matrix (ECM) hydrogel on day 5 ( E ), or onto a 2D monolayer of myotubes with a Geltrex undercoating on day 5 of differentiation ( F ). n=6–15 from N=2–3 independent biological replicates. Graphs display mean ± s.e.m. ( G ) Representative confocal images of YFP + (yellow) donor cells (DAPI: cyan) at 7 DPE engrafted in 2D, 3D with myotubes (day 5), 3D with myocytes (day 0), 3D with cellulose-reinforced ECM or with a 2D monolayer of myotubes. Cells are also labelled for Ki67 (magenta) and Pax7 (white) where Ki67 - Pax7 + cells are indicated with white arrows, Ki67 + Pax7 + with grey arrows, and Ki67 + Pax7 - with magenta arrows. Scale bar, 50 µm. Raw data available in . Figure 4—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

Techniques:

Journal: eLife

Article Title: The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( A ) Representative confocal images of two (Cell #1, left; Cell #2, right) mononucleated donor cells (DAPI: cyan; YFP: yellow) with M-cadherin (M-cad: white, white arrows) labelling restricted to the apical side. Cells are associated with myotubes that were visualized using the background signal (white dotted lines). Scale bars, 20 µm.

Article Snippet: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

Techniques:

Journal: eLife

Article Title: The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( A ) Representative confocal image of a mononuclear donor cell (DAPI: cyan, YFP: yellow) with neighbouring myotubes (Phalloidin: magenta) and N-cadherin (white) localized to the tip of the donor cell projection (white arrowhead). Scale bar, 20 µm. ( B ) Representative confocal images of a mononuclear donor cell (DAPI: cyan, YFP: yellow) at 1 day post-engraftment (DPE) (top) and 7 DPE (middle and bottom) expressing integrin α-7 (magenta) and M-cadherin (white). Middle inset image channels are separated to produce the bottom images to highlight the polarization of integrin α-7 and M-cadherin (white arrow) to basal and apical orientations, respectively (dotted lines). Scale bars, 20 µm. ( C ) Bar plot showing the percentage of mononuclear DAPI + YFP + cells with N-cadherin + cytoplasmic projections at 7 DPE. n=8 across N=3 independent biological replicates. Graph displays mean ± s.e.m. with individual technical replicates. ( D ) Bar plot showing the percentage of mononuclear DAPI + YFP + cells with polarized integrin α-7 (Iα7)/M-cadherin expression at 7 DPE. n=8 across N=3 independent biological replicates. Graph displays mean ± s.e.m. with individual technical replicates. Raw data available in . Figure 6—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

Techniques: Expressing