Journal: Function

Article Title: Satellite Cell Depletion Disrupts Transcriptional Coordination and Muscle Adaptation to Exercise

doi: 10.1093/function/zqaa033

Figure Lengend Snippet: PoWeR led to higher satellite cell content in the soleus and plantaris in SC+ mice. ( A ) Representative images of satellite cell IHC from the soleus across all groups showing laminin (green), nuclei (blue), and Pax7 (red; white arrows). ( B ) Satellite cell density in the soleus. ( C ) Satellite cell density in the plantaris. Scale bar = 50 µm. Data are represented as mean ± SEM. Statistical analysis: two-way ANOVA with Tukey’s multiple comparisons test. There was a main effect for PoWeR ( P < 0.0001) and satellite cell depletion ( P < 0.0001) in both the soleus and plantaris. There was a significant interaction between PoWeR and satellite cell content in the soleus ( P < 0.001) and plantaris ( P < 0.0001). * P < 0.05, ** P < 0.01.

Article Snippet: Samples were then incubated with antimouse biotin-conjugated secondary antibody against the Pax7 primary antibody (1:1000, 115-065-205, Jackson ImmunoResearch, West Grove, PA, USA) and antirabbit secondary for laminin (1:250, A11034, Alexa Fluor 488, Invitrogen, Carlsbad, CA, USA).

Techniques:

Journal: Cell reports

Article Title: In Situ Fixation Redefines Quiescence and Early Activation of Skeletal Muscle Stem Cells.

doi: 10.1016/j.celrep.2017.10.080

Figure Lengend Snippet: Figure 1. Isolation of MuSCs in a Fixed, G0-Arrested State (A) Graphical scheme of the in situ fixation protocol for MuSCs isolation and comparison to the standard protocol. A detailed description of the protocols is available in the Supplemental Information. BM, basement membrane; T0-SC, time-zero/quiescent MuSC; T3-SC, time 3 hr/activated MuSC. (B) FACS profiles of non-fixed (T3-SC) and fixed (T0-SC) GFP+ MuSCs from Tg:Pax7-nGFP muscle preparations. Sorted GFP+ cells are marked as red dots in all plots. SSC, side scatter, FSC, forward scatter. Values on the plots indicate mean percentage of sorted GFP+ cells of the total number of events, excluding small SSC/FSC and doublets; n = 5. (C) Proliferation of FACS-isolated T3-SCs and T0-SCs cultured for 48 hr and stained with EdU (24-hr chase); n = 3. EdU+ cells: 0% for T0-SCs and 74% ± 0.03% for T3-SCs. (D) Nascent RNA synthesis in FACS-isolated T3-SCs and T0-SCs cultured for 48 hr and incubated with labeled ethynyl uridine (EU) ribonucleoside for 2 hr; n = 3. Average of 80 nuclei counted per sample, 100% EU+ and EU for T3-SC and T0-SC, respectively. Dotted line delineates cell’s nucleus. (E) Morphology of T3 and T0 MuSCs immediately after the FACS. Cells were spun on Matrigel-coated slides, and membranes were stained with the lectin marker WGA (wheat germ agglutinin). Mean eccentricity of the cells was computationally calculated (Experimental Procedures). Data are reported as mean ± SD; n.d, not detected. Scale bars, 90 mm in (C), 20 mm in (D), 8 mm in (D, insets), and 10 mm in (E). See also Supplemental Information.

Article Snippet: The purity of the FACS-isolated cells was assessed with anti-PAX7 antibody (mouse monoclonal; Santa Cruz Biotechnology, #sc-81648) diluted 1:100 in BS and incubated for 16 hr at 37 C. For the membrane staining, WGA was used following manufacturer’s guidelines (Thermo Fisher Scientific, #W32464) on T0- or T3-SCs after a 15-min centrifugation (300 3 g, 4 C) on Matrigel (Corning, #354248)-coated 8-chamber slides (Sarstedt, #94.6140.802).

Techniques: Isolation, In Situ, Comparison, Membrane, Cell Culture, Staining, Incubation, Labeling, Marker

Journal: Cell reports

Article Title: In Situ Fixation Redefines Quiescence and Early Activation of Skeletal Muscle Stem Cells.

doi: 10.1016/j.celrep.2017.10.080

Figure Lengend Snippet: Figure 5. Histone H3 Modifications and DNA Methylation during Early MuSCs Activation (A) Left: H3K4me3 ChIP-seq averaged signal at promoters. Right: overlap between T0-SC and T3-SC genes with H3K4me3 peaks at promoters. TSS, tran- scription start site. (B) Left: H3K27ac ChIP-seq averaged signal at active distal enhancers (H3K27ac peaks outside gene body) from T0-SC and T3-SC samples. Right: overlap of genes that present at least one distal enhancer between T0-SCs and T3-SCs. (C) Examples of H3K4me3 and H3K27ac ChIP-seq tracks from T3 and T0 upregulated genes Fosl and Egr3. (D) Examples of H3K4me3 and H3K27ac ChIP-seq tracks from T3 and T0 downregulated genes Hes1 and Pax7. (E) H3K27me3 ChIP-seq averaged signal at promoters between T0-SCs, T3-SCs, and activated MuSCs from a published dataset (Liu et al., 2013). (F) Volcano plot representing genomic DNA methylation changes between T0-SCs and T3-SCs. Each dot represents one of 27,878 CpG clusters interrogated. Among all clusters (blue), those with a methylation change R 10% and an FDR < 0.01 were considered significantly different (red). FDR, false discovery rate (logarithmic scale).

Article Snippet: The purity of the FACS-isolated cells was assessed with anti-PAX7 antibody (mouse monoclonal; Santa Cruz Biotechnology, #sc-81648) diluted 1:100 in BS and incubated for 16 hr at 37 C. For the membrane staining, WGA was used following manufacturer’s guidelines (Thermo Fisher Scientific, #W32464) on T0- or T3-SCs after a 15-min centrifugation (300 3 g, 4 C) on Matrigel (Corning, #354248)-coated 8-chamber slides (Sarstedt, #94.6140.802).

Techniques: DNA Methylation Assay, Activation Assay, ChIP-sequencing, Methylation

Journal: Developmental biology

Article Title: CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos.

doi: 10.1016/j.ydbio.2024.11.013

Figure Lengend Snippet: Figure 3. One-color CRISPR-Cas13d-mediated PAX7 knockdown. (A) A representative 498

Article Snippet: 565 Jo urn al Pr e-p roo f KEY RESOURCES TABLE Reagent or resource Source Identifier Antibodies Mouse IgG1 anti-PAX7 Developmental Studies Hybridoma Bank Cat#: pax7 RRID: AB_528428 Goat IgG anti-GFP Rockland Cat#: 600-101-215 RRID: AB_218182 Rabbit igG anti-SOX10 Millipore/Sigma Cat#: HPA068898 RRID: AB_2686054 Donkey anti-Goat IgG Alexa Fluor 647 Invitrogen Cat#: A21447 Goat anti-Mouse IgG1 Alexa Fluor 647 Invitrogen Cat#: A21240 Goat anti-Rabbit IgG Alexa Fluor 568 Invitrogen Cat#: A11011 Chemicals, Peptides, and Recombinant Proteins DAPI Thermo Fisher Cat#: D1306 Fluoromount-G SouthernBiotech Cat#: 0100-01 Experimental Models: Organisms/Strains Gallus gallus Petaluma Farms (Petaluma, CA) Fertile Rhode Island Red eggs Oligonucleotides Morpholino: Control 5’-CCTCTTACCTCAGTTACAATTTATA GeneTools Morpholino: Pax7 5’-TCCGTGCGGAGCGGGTCACCCCC GeneTools; Basch et al., 2006 Primer: gRNA3 Forward: 5’-GTAATAGCGAAGAGGCCCGC Reverse: 5’-ATAGCGGCCGCTCATCTAGA This paper; IDT Custom DNA Oligos Recombinant DNA Plasmid: pCI-H2B-RFP Betancur et al., 2010 Plasmid: pCIG Megason and McMahon, 2002 Plasmid: pCAG-memRFP This paper; Addgene Addgene # 224564 Plasmid: pCAG-Cas13d-T2A-Citrine This paper; Addgene Addgene # 224565 Plasmid: pCAG-Cas13d-T2A-GFP(1-10) This paper; Addgene Addgene # 224566 Plasmid: pCAG-memRFP-3xControlgRNA This paper; Addgene Addgene # 224567 Plasmid: pCAG-nucGFP11-3xControlgRNA This paper; Addgene Addgene # 224568 Plasmid: pCAG-3xGFP11-mem-3xControlgRNA This paper; Addgene Addgene # 224583 Plasmid: pCAG-memRFP-3xPax7gRNA This paper; Addgene Addgene # 224569 Plasmid: pCAG-nucGFP11-3xPax7gRNA This paper; Addgene Addgene # 224570 Plasmid: pCAG-3xGFP11-mem-3xPax7gRNA This paper; Addgene Addgene # 224571 Software and Algorithms Fiji Schindelin et al., 2012 RRID:SCR_002285 Zen 2 Blue Zeiss Photoshop CC Adobe Prism 10 GraphPad Jo urn al Pr e-p roo f HIGHLIGHTS • A CRISPR-Cas13d system is functional in chick embryos using a two-plasmid approach • CRISPR-Cas13d-mediated knockdown of PAX7 leads to decreased expression and function • CRISPR-Cas13d and morpholino yield comparable effects for PAX7 knockdown Jo urn al Pr e-p roo f

Techniques: CRISPR, Knockdown

Journal: Developmental biology

Article Title: CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos.

doi: 10.1016/j.ydbio.2024.11.013

Figure Lengend Snippet: Figure 4. A comparison of morpholino- and CRISPR-Cas13d-mediated PAX7 knockdown 512

Article Snippet: 565 Jo urn al Pr e-p roo f KEY RESOURCES TABLE Reagent or resource Source Identifier Antibodies Mouse IgG1 anti-PAX7 Developmental Studies Hybridoma Bank Cat#: pax7 RRID: AB_528428 Goat IgG anti-GFP Rockland Cat#: 600-101-215 RRID: AB_218182 Rabbit igG anti-SOX10 Millipore/Sigma Cat#: HPA068898 RRID: AB_2686054 Donkey anti-Goat IgG Alexa Fluor 647 Invitrogen Cat#: A21447 Goat anti-Mouse IgG1 Alexa Fluor 647 Invitrogen Cat#: A21240 Goat anti-Rabbit IgG Alexa Fluor 568 Invitrogen Cat#: A11011 Chemicals, Peptides, and Recombinant Proteins DAPI Thermo Fisher Cat#: D1306 Fluoromount-G SouthernBiotech Cat#: 0100-01 Experimental Models: Organisms/Strains Gallus gallus Petaluma Farms (Petaluma, CA) Fertile Rhode Island Red eggs Oligonucleotides Morpholino: Control 5’-CCTCTTACCTCAGTTACAATTTATA GeneTools Morpholino: Pax7 5’-TCCGTGCGGAGCGGGTCACCCCC GeneTools; Basch et al., 2006 Primer: gRNA3 Forward: 5’-GTAATAGCGAAGAGGCCCGC Reverse: 5’-ATAGCGGCCGCTCATCTAGA This paper; IDT Custom DNA Oligos Recombinant DNA Plasmid: pCI-H2B-RFP Betancur et al., 2010 Plasmid: pCIG Megason and McMahon, 2002 Plasmid: pCAG-memRFP This paper; Addgene Addgene # 224564 Plasmid: pCAG-Cas13d-T2A-Citrine This paper; Addgene Addgene # 224565 Plasmid: pCAG-Cas13d-T2A-GFP(1-10) This paper; Addgene Addgene # 224566 Plasmid: pCAG-memRFP-3xControlgRNA This paper; Addgene Addgene # 224567 Plasmid: pCAG-nucGFP11-3xControlgRNA This paper; Addgene Addgene # 224568 Plasmid: pCAG-3xGFP11-mem-3xControlgRNA This paper; Addgene Addgene # 224583 Plasmid: pCAG-memRFP-3xPax7gRNA This paper; Addgene Addgene # 224569 Plasmid: pCAG-nucGFP11-3xPax7gRNA This paper; Addgene Addgene # 224570 Plasmid: pCAG-3xGFP11-mem-3xPax7gRNA This paper; Addgene Addgene # 224571 Software and Algorithms Fiji Schindelin et al., 2012 RRID:SCR_002285 Zen 2 Blue Zeiss Photoshop CC Adobe Prism 10 GraphPad Jo urn al Pr e-p roo f HIGHLIGHTS • A CRISPR-Cas13d system is functional in chick embryos using a two-plasmid approach • CRISPR-Cas13d-mediated knockdown of PAX7 leads to decreased expression and function • CRISPR-Cas13d and morpholino yield comparable effects for PAX7 knockdown Jo urn al Pr e-p roo f

Techniques: Comparison, CRISPR, Knockdown

Journal: Food Science of Animal Resources

Article Title: The L-Ascorbic Acid Increases Proliferation and Differentiation of Yanbian Cattle Skeletal Muscle Satellite Cells by Activating the Akt/mTOR/P70S6K Signaling Pathway

doi: 10.5851/kosfa.2024.e50

Figure Lengend Snippet: Primer sequences

Article Snippet: BSCs were then immunofluorescently stained with an antibody against Pax7 (mouse anti-Pax7,1:200, Proteintech), a key marker factor for BSCs.

Techniques: Sequencing

Journal: Food Science of Animal Resources

Article Title: The L-Ascorbic Acid Increases Proliferation and Differentiation of Yanbian Cattle Skeletal Muscle Satellite Cells by Activating the Akt/mTOR/P70S6K Signaling Pathway

doi: 10.5851/kosfa.2024.e50

Figure Lengend Snippet: Cell viability was measured using CCK-8 after treatment of BSCs with different concentrations (50, 100, 200, 400, and 800 μM) of AA for 24, 48, and 72 h (A). The percentage of EdU-positive cells was detected by EdU staining under fluorescence microscopy after 48 h treatment of BSCs using different concentrations of AA (B; scale bar=200 μm). The histogram shows the statistics of positive cells after 48 h treatment of BSCs using different concentrations of AA (C). The mRNA expression levels of the gene encoding the transporter protein SLC23A2 after 48 h treatment of BSCs with different concentrations (100, 200, and 400 μM) of AA by real-time quantitative PCR (RT-qPCR). The expression levels of Ki67, proliferating cell nuclear antigen (PCNA), cell cycle protein-dependent kinase 1 (CDK1) and CDK2 proliferation-related genes were measured by RT-qPCR at different concentrations (100, 200, and 400 μM) of AA treated BSCs for 48 h (D). The expression levels of proliferation marker paired box 7 (Pax7) were measured by RT-qPCR and Western blotting after treatment of BSCs with different concentrations of AA for 48 h (E, F). All experiments were repeated ≥3 times and compared using t-test. Data are presented as mean±SEM. a–e Significant differences between treatment groups are indicated by different letters (p<0.05), and the same letter or no letter marked indicates no significant difference between groups (p>0.05).

Article Snippet: BSCs were then immunofluorescently stained with an antibody against Pax7 (mouse anti-Pax7,1:200, Proteintech), a key marker factor for BSCs.

Techniques: CCK-8 Assay, Staining, Fluorescence, Microscopy, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Western Blot

Journal: Food Science of Animal Resources

Article Title: The L-Ascorbic Acid Increases Proliferation and Differentiation of Yanbian Cattle Skeletal Muscle Satellite Cells by Activating the Akt/mTOR/P70S6K Signaling Pathway

doi: 10.5851/kosfa.2024.e50

Figure Lengend Snippet: Cell viability was measured by CCK-8 after 48 h treatment of BSCs using different concentrations (10, 20, 100, 200, and 400 nM) of rapamycin (A). Detection of EdU positive cell rates under fluorescent microscopy using EdU staining (B; scale bar=200 μm). The bar graph shows the differences between the treatment groups (C). The mRNA expression levels of proliferation-related genes paired box 7 (Pax7), Ki67, and proliferating cell nuclear antigen (PCNA) were detected using real-time quantitative PCR (D). The mTOR pathway was used to detect the level of Pax7 protein expression under the regulation of L-ascorbic acid (AA) using Western blotting (E). a–d Significant differences between treatment groups are indicated by different letters (p<0.05), and the same letter or no letter marked indicates no significant difference between groups (p>0.05).

Article Snippet: BSCs were then immunofluorescently stained with an antibody against Pax7 (mouse anti-Pax7,1:200, Proteintech), a key marker factor for BSCs.

Techniques: CCK-8 Assay, Microscopy, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot