Journal: International Journal of Molecular Sciences

Article Title: Interleukin-6 Induces Myogenic Differentiation via JAK2-STAT3 Signaling in Mouse C2C12 Myoblast Cell Line and Primary Human Myoblasts

doi: 10.3390/ijms20215273

Figure Lengend Snippet: Primary antibodies utilized for western blotting and immunocytochemistry.

Article Snippet: MyoD , Santa Cruz , Mouse , SC32758.

Techniques: Western Blot, Immunocytochemistry

Journal: bioRxiv

Article Title: Canonical Wnt signaling regulates soft palate development through mediating ciliary homeostasis

doi: 10.1101/2022.08.03.502715

Figure Lengend Snippet: ( A-D ) Hematoxylin and eosin staining at the level of the levator veli palatini (LVP) in control (A-B) and Osr2-Cre;β-catenin fl/fl mice ( C-D ) at E18.5. Black asterisks in C and D identify the cleft palate in Osr2-Cre;β-catenin fl/fl . (E-H) MHC (green) immunofluorescence in control (E-F) and Osr2-Cre;β-catenin fl/fl mice ( G-H ) at E18.5. White asterisks in G and H identify the cleft palate in Osr2-Cre;β-catenin fl/fl . (I-L) MyoD (green) immunofluorescence in control (I-J) and Osr2-Cre;β-catenin fl/fl mice (K-L) at E13.5. (M-P) Myod1 (green) in situ RNAScope hybridization in control (M-N) and Osr2-Cre;β-catenin fl/fl mice ( O-P ) at E14.0 (M, O) and E14.5 (N, P). White asterisk in P identifies the cleft palate in Osr2-Cre;β-catenin fl/fl . Schematic drawing of the mouse head in the top panel depicts the position and angle of sectioning. Black boxes in A and C show approximate locations of higher magnification images in B and D, respectively. White boxes in E, G, I, and K show approximate locations of higher magnification images in F, H, J, M, N, L, O, and P. Red dotted boxes in J, L, and M-P show the position of the insets in their lower left corners. Black dotted lines in A and C indicate palatal shelves. White dotted lines in E, G, I, and K indicate palatal shelves. White dashed lines outline the LVP muscle region stained with MHC, MyoD, or Myod1 in F, J, L, and M-P. Scale bar in A indicates 200 µm in A, and C; scale bar in B indicates 100 µm in B, and D; scale bar in E indicates 100 µm in E, G, I and K; scale bar in F indicates 100 µm in F, H, N, and P; scale bar in J indicates 100 µm in J, L, M, and O, respectively.

Article Snippet: Primary antibodies used in this study were as follows: rabbit monoclonal active β-catenin (Cell Signaling, 19807S, 1:100 with TSA), mouse monoclonal, myosin heavy chain, MHC (DSHB, MF20, 1:10), rabbit polyclonal anti-phospho-histone H3, pH3 (Sigma Aldrich, 06-570, 1:100), rabbit monoclonal cleaved caspase-3 (Cell Signaling, 9664S, 1:100), rabbit monoclonal MyoD (Abcam, ab133627, 1:200 with TSA), rabbit polyclonal, γ-tubulin (Sigma Aldrich, T5192, 1:100), rabbit polyclonal Arl13b (Proteintech, 17711-1-AP, 1:100), rabbit polyclonal red fluorescein protein, RFP (Rockland, 600-401-379, 1:500 with TSA), rat monoclonal BrdU (Abcam, ab6326, 1:100).

Techniques: Staining, Control, Immunofluorescence, In Situ, RNAscope, Hybridization

Journal: bioRxiv

Article Title: Canonical Wnt signaling regulates soft palate development through mediating ciliary homeostasis

doi: 10.1101/2022.08.03.502715

Figure Lengend Snippet: (A, D) DAPI staining of control (A) and Osr2-Cre;β-catenin fl/fl (D) sections at the level of the LVP at E13.5. (B-C, E-F) MyoD (green) and pH3 (red) (B, E), or MyoD (green) and caspase3 (red) immunohistofluorescence staining (C, F) in control (B-C) and Osr2-Cre;β-catenin fl/fl mice (E-F). (G-H, J-K) BrdU (green) and pH3 (red) immunohistofluorescence staining at E14.0 in control (G-H) and Osr2-Cre;β-catenin fl/fl mice (J-K). (I) Quantification of the percentage of pH3 positive cells. **p-value = 0.0031. (L) Quantification of percentage of BrdU+ pH3+ double positive cells out of total BrdU+ cells. **p-value = 0.0091. Schematic drawing of the mouse head in the top panel depicts the position and angle of sectioning. Schematic drawing at the bottom indicates injection of pregnant mouse with BrdU followed by collection of embryos 2 hours later. White boxes in A, D, G, and J show approximate location of higher magnification images in B-C, E-F, H, and K, respectively. Yellow dotted boxes in H and K show the position of the insets in their lower left corners. White arrows in B and E point to the positive signal. White arrowheads in H and K point to positive cells. Yellow arrowheads in H and K point to double positive cells. Scale bar in A indicates 100 µm in A, D, G, and J; scale bar in B indicates 50 µm in B-C, E-F, H, and K.

Article Snippet: Primary antibodies used in this study were as follows: rabbit monoclonal active β-catenin (Cell Signaling, 19807S, 1:100 with TSA), mouse monoclonal, myosin heavy chain, MHC (DSHB, MF20, 1:10), rabbit polyclonal anti-phospho-histone H3, pH3 (Sigma Aldrich, 06-570, 1:100), rabbit monoclonal cleaved caspase-3 (Cell Signaling, 9664S, 1:100), rabbit monoclonal MyoD (Abcam, ab133627, 1:200 with TSA), rabbit polyclonal, γ-tubulin (Sigma Aldrich, T5192, 1:100), rabbit polyclonal Arl13b (Proteintech, 17711-1-AP, 1:100), rabbit polyclonal red fluorescein protein, RFP (Rockland, 600-401-379, 1:500 with TSA), rat monoclonal BrdU (Abcam, ab6326, 1:100).

Techniques: Staining, Control, Immunohistofluorescence, Injection

Journal: bioRxiv

Article Title: Canonical Wnt signaling regulates soft palate development through mediating ciliary homeostasis

doi: 10.1101/2022.08.03.502715

Figure Lengend Snippet: (A-C) RNAseq analysis of soft palatal samples from control (blue) and Osr2-Cre;β-catenin fl/fl mice (red) collected at E14.0 depicted with heat map (A), PCA plot (B), and volcano plot (C). RNAseq analysis was performed under the following conditions: p-value ≤ 0.05, fold change ≤-1.5 or ≥ 1.5. A total of 2122 differentially expressed genes were identified, out of which 1810 were upregulated and 622 downregulated. (D) The most changed GO terms from the Partek Flow analysis point to the changes in muscle (green) and cilium development (red). (E) Schematic drawing at the level of the levator veli palatini level (LVP). (F) γ-tubulin quantification indicates the percentage of ciliated cells. **p-value = 0.0012. (G-J) γ-tubulin (red) and MyoD (green) immunohistofluorescence staining at E13.5 in control (G-H) and Osr2-Cre;β-catenin fl/fl mice (I-J). (K-N) Arl13b (red) and MyoD (green) immunohistofluorescence staining at E13.5 in control (K-L) and Osr2-Cre;β-catenin fl/fl mice (M-N). (O-R) Ttll3 (red) in situ RNAScope hybridization and MyoD (green) immunohistofluorescence staining in control (O-P) and Osr2-Cre;β-catenin fl/fl mice (Q-R). (S-V) Ift88 (red) and Myod1 (green) in situ RNAScope hybridization in control (S-T) and Osr2-Cre;β-catenin fl/fl mice (U-V). Schematic drawing of the mouse head in the top panel depicts the position and angle of sectioning. Black box in E shows approximate location of images in G, I, K, M, O, Q, S, and U. White boxes in G, I, K, M, O, Q, S, and U are showing a higher magnification in H, J, L, N, P, R, T, and V, respectively. White arrows in H, J, L, N, P, R, T, and V point to the positive signal. Scale bar in G indicates 50 µm in G, I, K, M, O, Q, S, and U; scale bar in H indicates 20 µm in H, J, L, N, P, R, T, and V.

Article Snippet: Primary antibodies used in this study were as follows: rabbit monoclonal active β-catenin (Cell Signaling, 19807S, 1:100 with TSA), mouse monoclonal, myosin heavy chain, MHC (DSHB, MF20, 1:10), rabbit polyclonal anti-phospho-histone H3, pH3 (Sigma Aldrich, 06-570, 1:100), rabbit monoclonal cleaved caspase-3 (Cell Signaling, 9664S, 1:100), rabbit monoclonal MyoD (Abcam, ab133627, 1:200 with TSA), rabbit polyclonal, γ-tubulin (Sigma Aldrich, T5192, 1:100), rabbit polyclonal Arl13b (Proteintech, 17711-1-AP, 1:100), rabbit polyclonal red fluorescein protein, RFP (Rockland, 600-401-379, 1:500 with TSA), rat monoclonal BrdU (Abcam, ab6326, 1:100).

Techniques: Control, Immunohistofluorescence, Staining, In Situ, RNAscope, Hybridization

Journal: bioRxiv

Article Title: Canonical Wnt signaling regulates soft palate development through mediating ciliary homeostasis

doi: 10.1101/2022.08.03.502715

Figure Lengend Snippet: (A) Schematic drawings of primary cilia focusing on ciliary structure (basal body and ciliary axoneme), intraflagellar transport and posttranslational modifications. (B-E) Ttll6 (red) and Myod1 (green) in situ RNAScope hybridization in control (B-C) and Osr2-Cre;β-catenin fl/fl mice (D-E). Schematic drawing of the mouse head in the top panel depicts the position and angle of sectioning. Boxes in B and D show approximate location of images in C and E. White arrows in C and E point to positive signals. Scale bar in B indicates 50 µm in B, and D; scale bar in C indicates 20 µm in C and E.

Article Snippet: Primary antibodies used in this study were as follows: rabbit monoclonal active β-catenin (Cell Signaling, 19807S, 1:100 with TSA), mouse monoclonal, myosin heavy chain, MHC (DSHB, MF20, 1:10), rabbit polyclonal anti-phospho-histone H3, pH3 (Sigma Aldrich, 06-570, 1:100), rabbit monoclonal cleaved caspase-3 (Cell Signaling, 9664S, 1:100), rabbit monoclonal MyoD (Abcam, ab133627, 1:200 with TSA), rabbit polyclonal, γ-tubulin (Sigma Aldrich, T5192, 1:100), rabbit polyclonal Arl13b (Proteintech, 17711-1-AP, 1:100), rabbit polyclonal red fluorescein protein, RFP (Rockland, 600-401-379, 1:500 with TSA), rat monoclonal BrdU (Abcam, ab6326, 1:100).

Techniques: In Situ, RNAscope, Hybridization, Control

Journal: bioRxiv

Article Title: Canonical Wnt signaling regulates soft palate development through mediating ciliary homeostasis

doi: 10.1101/2022.08.03.502715

Figure Lengend Snippet: (A) CellChat analysis of E13.5 soft palatal scRNAseq data showing a heatmap visualizing the contribution of the outgoing signaling patterns. 0, midline mesenchymal cells; 1, CNC-derived progenitor cells; 2, perimysial cells; 3, osteogenic cells; 4, perimysial cells; 5, chondrogenic cells; 6, mitotic cells; 7, neuronal cells; 8, epithelial cells; 9, perimysial cells; 10, midline mesenchymal cells; 11, glial cells; 12, red blood cells; 13, chondrogenic cells; 14, myogenic cells; 15, neuronal cells; 16, glial cells; 17, myeloid cells. The red box highlights Notch signaling and black boxes mark perimysial clusters (2, 4, 9). (B) Notch signaling activity is shown in the direction of perimysial to myogenic cells (2→14, 4→14, and 9→14). Red dotted box highlights Dlk1/Notch3 ligand and receptor combination. (C-F) Dlk1 (red) in situ RNAScope hybridization and MyoD (green) immunohistochemistry staining at E13.5 in control (C-D) and Osr2-Cre;β-catenin fl/fl mice (E-F). (G-H) qPCR analysis. Efficiency of Ttll3 siRNA. *p-value = 0.0297 (G). Increased expression of Dlk1 after Ttll3 siRNA treatment. *p-value = 0.0477 (H). Schematic drawing of the mouse head in the top panel depicts the position and angle of sectioning. White boxes in C and E show approximate location of higher magnification images in D and F, respectively. White dotted boxes in D and F show the position of the insets in their lower left corners. White dotted lines indicate palatal shelves in C and E. White arrow in D points to positive signal. Asterisk in F shows a lack of positive signal. Scale bar in C indicates 100 µm in C and E; scale bar in D indicates 50 µm in D and F.

Article Snippet: Primary antibodies used in this study were as follows: rabbit monoclonal active β-catenin (Cell Signaling, 19807S, 1:100 with TSA), mouse monoclonal, myosin heavy chain, MHC (DSHB, MF20, 1:10), rabbit polyclonal anti-phospho-histone H3, pH3 (Sigma Aldrich, 06-570, 1:100), rabbit monoclonal cleaved caspase-3 (Cell Signaling, 9664S, 1:100), rabbit monoclonal MyoD (Abcam, ab133627, 1:200 with TSA), rabbit polyclonal, γ-tubulin (Sigma Aldrich, T5192, 1:100), rabbit polyclonal Arl13b (Proteintech, 17711-1-AP, 1:100), rabbit polyclonal red fluorescein protein, RFP (Rockland, 600-401-379, 1:500 with TSA), rat monoclonal BrdU (Abcam, ab6326, 1:100).

Techniques: Derivative Assay, Activity Assay, In Situ, RNAscope, Hybridization, Immunohistochemistry, Staining, Control, Expressing

Journal: Oncotarget

Article Title: NDRG4 promotes myogenesis via Akt/CREB activation

doi: 10.18632/oncotarget.21591

Figure Lengend Snippet: (A) qRT-PCR analysis of the expression of NDRG4 in C2C12 cells at days 0, 2, and 4 after differentiation. (B) Western blot analysis of the NDRG4 protein in C2C12 cells at days 0, 2, and 4 after differentiation. Myogenin (MyoG) and MyHC are two marker genes of muscle differentiation. (C) qRT-PCR results showing the expression of the NDRG4 , myogenic differentiation factor ( MyoD ), and MyoG genes during muscle regeneration. Hindlimb muscle was subjected to cardiotoxin (CTX) injection and harvested on the indicated days after injury for RNA analysis. GAPDH was used as an internal control. Values were normalized to GAPDH. Data are presented as means ± standard error of the mean (n = 3). Asterisks above columns represent significant differences among groups ( ** P < 0.01).

Article Snippet: The membranes were blocked with 5% non-fat milk for 2 h and incubated overnight at 4°C with primary antibodies against NDRG4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-166,917, 1:200 dilution), MyoG (Santa Cruz Biotechnology; sc-12,732, 1:200 dilution), MyHC (Santa Cruz Biotechnology; sc-376,157, 1:3000 dilution), CREB (Cell Signaling Technology, Danvers, MA, USA; 48H2, 1:2000 dilution), GAPDH (Boster, China; BM0627, 1:1000 dilution), MyoD (Santa Cruz Biotechnology; sc-377,186, 1:200 dilution), Akt (Cell Signaling Technology, Danvers, MA, USA; 40D4, 1:2000 dilution), pAkt (Cell Signaling Technology, Danvers, MA, USA; D9E, 1:1000 dilution), carboxyl-terminal modulator protein (CTMP) (Santa Cruz Biotechnology, CA, sc-390,353 1:200 dilution), PTEN (Cell Signaling Technology, Danvers, MA, USA; Y184, 1:1000 dilution), and phospho-CREB at ser133 rabbit monoclonal (Cell Signaling Technology, Danvers, MA, USA; 87G3), followed by incubation with goat anti-mouse IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology; sc-2005, 1:3000 dilution) and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology; sc-2004, 1:3000 dilution) secondary antibodies for 1 h at room temperature.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Marker, Injection, Control

Journal: Oncotarget

Article Title: NDRG4 promotes myogenesis via Akt/CREB activation

doi: 10.18632/oncotarget.21591

Figure Lengend Snippet: (A) Western blot results of NDRG4, MyoD, MyoG, and MyHC protein expression levels after NDRG4 knockdown at 0 days (D0), 2 days (D2), and 4 days (D4). (B) Relative protein expression levels represented by the ratio of detected protein to the GAPDH protein expression level after NDRG4 knockdown at D0, D2, and D4. The quantifications of Western blot data are presented as means ± standard deviation (SD; n = 3). (C) Immunofluorescence results of NDRG4 and MyHC protein expression levels after NDRG4 knockdown. Transfected C2C12 myoblasts were differentiated for 5 days, and stained with anti-NDRG4 (green) and anti-MyHC antibodies (red), and 4’,6-diamidino-2-phenylindole (DAPI; blue), and imaged by fluorescence microscopy. Bars, 200 μm. (D) The quantification of myotube formation is shown in the panel.

Article Snippet: The membranes were blocked with 5% non-fat milk for 2 h and incubated overnight at 4°C with primary antibodies against NDRG4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-166,917, 1:200 dilution), MyoG (Santa Cruz Biotechnology; sc-12,732, 1:200 dilution), MyHC (Santa Cruz Biotechnology; sc-376,157, 1:3000 dilution), CREB (Cell Signaling Technology, Danvers, MA, USA; 48H2, 1:2000 dilution), GAPDH (Boster, China; BM0627, 1:1000 dilution), MyoD (Santa Cruz Biotechnology; sc-377,186, 1:200 dilution), Akt (Cell Signaling Technology, Danvers, MA, USA; 40D4, 1:2000 dilution), pAkt (Cell Signaling Technology, Danvers, MA, USA; D9E, 1:1000 dilution), carboxyl-terminal modulator protein (CTMP) (Santa Cruz Biotechnology, CA, sc-390,353 1:200 dilution), PTEN (Cell Signaling Technology, Danvers, MA, USA; Y184, 1:1000 dilution), and phospho-CREB at ser133 rabbit monoclonal (Cell Signaling Technology, Danvers, MA, USA; 87G3), followed by incubation with goat anti-mouse IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology; sc-2005, 1:3000 dilution) and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology; sc-2004, 1:3000 dilution) secondary antibodies for 1 h at room temperature.

Techniques: Western Blot, Expressing, Knockdown, Standard Deviation, Immunofluorescence, Transfection, Staining, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: NDRG4 promotes myogenesis via Akt/CREB activation

doi: 10.18632/oncotarget.21591

Figure Lengend Snippet: (A) Western blot results of NDRG4, MyoD, MyoG, and MyHC protein expression levels after NDRG4 overexpression at D0, D2, and D4. (B) Relative protein expression levels represented by the ratio of detected protein to GAPDH protein expression level after NDRG4 overexpression at D0, D2, and D4. (C) Immunofluorescence results of NDRG4 and MyHC protein expression levels after NDRG4 overexpression. Transfected C2C12 myoblasts were differentiated for 3 days, stained with anti-NDRG4 (green) and anti-MyHC antibodies (red), and DAPI (blue), and imaged by fluorescence microscopy. Bars, 200 μm. (D) The quantification of myotube formation is shown in the panel.

Article Snippet: The membranes were blocked with 5% non-fat milk for 2 h and incubated overnight at 4°C with primary antibodies against NDRG4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-166,917, 1:200 dilution), MyoG (Santa Cruz Biotechnology; sc-12,732, 1:200 dilution), MyHC (Santa Cruz Biotechnology; sc-376,157, 1:3000 dilution), CREB (Cell Signaling Technology, Danvers, MA, USA; 48H2, 1:2000 dilution), GAPDH (Boster, China; BM0627, 1:1000 dilution), MyoD (Santa Cruz Biotechnology; sc-377,186, 1:200 dilution), Akt (Cell Signaling Technology, Danvers, MA, USA; 40D4, 1:2000 dilution), pAkt (Cell Signaling Technology, Danvers, MA, USA; D9E, 1:1000 dilution), carboxyl-terminal modulator protein (CTMP) (Santa Cruz Biotechnology, CA, sc-390,353 1:200 dilution), PTEN (Cell Signaling Technology, Danvers, MA, USA; Y184, 1:1000 dilution), and phospho-CREB at ser133 rabbit monoclonal (Cell Signaling Technology, Danvers, MA, USA; 87G3), followed by incubation with goat anti-mouse IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology; sc-2005, 1:3000 dilution) and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology; sc-2004, 1:3000 dilution) secondary antibodies for 1 h at room temperature.

Techniques: Western Blot, Expressing, Over Expression, Immunofluorescence, Transfection, Staining, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Neurofibromin 1 controls metabolic balance and Notch-dependent quiescence of murine juvenile myogenic progenitors

doi: 10.1038/s41467-024-45618-z

Figure Lengend Snippet: a Ki67 + /Pax7 + cells quantification relative to all Pax7 + cells in TA muscles of control and Nf1 Myf5 mice at indicated time points. p, postnatal day ( n = 3 animals per genotype; p -values shown). b Representative immunolabeling images of Pax7 (red), Ki67 (green), and DAPI (nuclei, blue) of p21 muscle sections of control or Nf1 Myf5 mice. Arrows indicate Pax7 + /Ki67 + cells ( n = 3 animals per genotype). c Cytospin of FACS-isolated p14 MPs from control or Nf1 Myf5 mice labeled for Pax7 (green), Ki67 (red), and DAPI (nuclei, blue). Quantification of Ki67 + /Pax7 + cells relative to all Pax7 + cells shown right ( n = 3 animals per genotype; p-value shown). d Quantification of anti-Pax7 relative fluorescence intensity (RFI) on images as in ( c ). Data range is shown as violin plot with median and interquartile range, means of biological replicates are shown as dots ( n = 3 animals per genotype; p -value shown). e Cytospin of FACS-isolated p14 MPs from control or Nf1 Myf5 mice labeled for Pax7 (green), MyoD (red), and DAPI (nuclei, blue). Quantification of MyoD + /Pax7 + cells (right) ( n = 3 animals per genotype; p -value shown). f In vitro differentiation of FACS-isolated p14 MPs from control or Nf1 Myf5 mice after 2 d differentiation stained for Myosin (Mf20, green), MyoD (red) and DAPI (nuclei, blue). Quantification of MyoD + nuclei within Mf20+ myotubes relative to all MyoD + nuclei (right) ( n = 3 animals per genotype; p -value shown). g Left: Representative images of single fibers isolated from 15-week EDL muscles stained for MyHC-2B (red) and DAPI (nuclei, blue). Boxed region shown as magnification. Right: Quantification of nuclei per myofiber and myonuclear domain (cell volume/number of nuclei); pL picoliter, ( n = 3 animals per genotype; p -values shown). h Pax7 + cell quantification on sections of TA muscles of control or Nf1 Myf5 mice at indicated time points ( n = 3 animals per genotype; p -values shown). Data are mean ± SEM; P -value calculated by two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies and dilutions used were: Mouse anti-Pax7 (DSHB # Pax7; 1:25), Goat anti-Pax7 (provided by C. Birchmeier; 1:100), Rabbit anti-Ki67 (Abcam # ab16667; 1:500), Mouse anti-Ki67 (BD Biosciences #550609; 1:500), Rabbit anti-MyoD (Cell Signaling Technology #13812; 1:300), Mouse anti-MyoD (BD Biosciences # 554130; 1:500), Mouse anti-MF20 (DSHB # MF20; 1:50), Anti-Myosin Skeletal, Fast (Sigma–Aldrich # M1570; 1:500), Rabbit anti- pErk1/2 (Cell Signaling Technology # 9101; 1:300), Rabbit anti-phosphor (s235/236)-S6 (Cell Signaling Technology # 4858; 1:300), Goat anti-Desmin (R&D Systems # AF3844; 1:500), Anti-trimethyl-Histone H3 (Lys27) (Millipore # 07-449; 1:500), anti-acetyl-Histone H4 (Lys16) (Millipore # 07-329; 1:500) (see also Supplementary Table ).

Techniques: Muscles, Immunolabeling, Isolation, Labeling, Fluorescence, In Vitro, Staining

Journal: Nature Communications

Article Title: Neurofibromin 1 controls metabolic balance and Notch-dependent quiescence of murine juvenile myogenic progenitors

doi: 10.1038/s41467-024-45618-z

Figure Lengend Snippet: a RT-qPCR of Notch pathway component and target genes in RNA extracted from freshly FACS-isolated control and Nf1 Myf5 p7 MPs ( n = 3 animals per genotype; each dot represents the mean of three technical replicates from one biological replicate; p -values shown). b RT-qPCR for Notch targets on FACS-isolated control or Nf1 Myf5 p14 MPs cultured on Matrigel without coating or Jagged-1 coating for 48 h ( n = 3 independent experiments from 3 animals per genotype; p -values shown). c FACS-isolated control or Nf1 Myf5 p14 MPs cultured on Matrigel without coating or Jagged-1 coating for 48 h stained for Pax7 (green) and Ki67 (red). d Ki67 + cell quantification among Pax7 + cells on image data as in ( c ) ( n = 3 animals per genotype; p -values shown). e Anti-Pax7 relative fluorescence intensity (RFI) quantification on image data as in ( c ). Data range is shown as violin plot with median and interquartile range, means of biological replicates are shown as dots ( n = 3 animals per genotype; p -values shown). f Immunolabeling for MyoD (red) on FACS-isolated control or Nf1 Myf5 p14 MPs cultured on Matrigel w/o coating or Jagged-1 coating for 48 h. g Quantification of MyoD + cells / total cells on image data as in ( f ) ( n = 3 animals per genotype; p -values shown). h Quantification of anti-MyoD relative fluorescence intensity (RFI) on image data as in ( f ). Data range is shown as violin plot with median and interquartile range, means of biological replicates are shown as dots ( n = 3 animals per genotype; p -values shown). i GSEA on RNA-Seq data from control and Nf1 Myf5 p7 MPs for “nitric oxide stimulates guanylate cyclase”. j RT-qPCR for Notch targets Pax7 , Hes1 and Hey1 on FACS-isolated control or Nf1 Myf5 p14 MPs. MPs were cultured on Matrigel without coating or with Jagged-1 coating for 48 h, with or without Mek inhibitor UO126 or pan-NOS inhibitor L-NAME. Bars show fold-changes of Nf1 Myf5 MPs relative to control MPs, control MPs were set as 1 ( n = 3 independent experiments from 3 animals per genotype; p -values shown). Data are mean ± SEM; P -value calculated by two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies and dilutions used were: Mouse anti-Pax7 (DSHB # Pax7; 1:25), Goat anti-Pax7 (provided by C. Birchmeier; 1:100), Rabbit anti-Ki67 (Abcam # ab16667; 1:500), Mouse anti-Ki67 (BD Biosciences #550609; 1:500), Rabbit anti-MyoD (Cell Signaling Technology #13812; 1:300), Mouse anti-MyoD (BD Biosciences # 554130; 1:500), Mouse anti-MF20 (DSHB # MF20; 1:50), Anti-Myosin Skeletal, Fast (Sigma–Aldrich # M1570; 1:500), Rabbit anti- pErk1/2 (Cell Signaling Technology # 9101; 1:300), Rabbit anti-phosphor (s235/236)-S6 (Cell Signaling Technology # 4858; 1:300), Goat anti-Desmin (R&D Systems # AF3844; 1:500), Anti-trimethyl-Histone H3 (Lys27) (Millipore # 07-449; 1:500), anti-acetyl-Histone H4 (Lys16) (Millipore # 07-329; 1:500) (see also Supplementary Table ).

Techniques: Quantitative RT-PCR, Isolation, Cell Culture, Staining, Fluorescence, Immunolabeling, RNA Sequencing Assay