iglon4  (St Johns Laboratory)

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 1. IgLON expressions in murine C2C12 myoblasts and primary MSCs during myogenic differentiation, and IgLON4 protein expression in regenerating mouse muscle: (A) mRNA and protein expressions of IgLONs (IgLON1 to 5) in C2C12 myoblasts on differentiation day 2 (DD2) as determined by real-time RT-PCR, and protein expressions of IgLON4 and 5 on DD2 as determined by Western blot. Locations of IgLON4 protein were determined by immunocytochemistry. (B) mRNA expressions of IgLONs in mouse primary MSCs on DD2 as determined by real-time RT-PCR, and protein expressions of IgLON4 and 5 as determined by Western blot analysis. (C) Expressions of IgLON4 in control and CTX-injected mouse gastrocnemius muscles during regeneration (differentia- tion days 3 and 7), as determined by immunofluorescence; muscle was collected and embedded with paraffin and then stained with IgLON4 and laminin (Green: IgLON4, Red: Laminin, Blue: Nucleus). (D) Expressions of IgLON4 in control and CTX-injected mouse gastrocnemius muscles during regen- eration (differentiation day 7), as determined by H&E staining and immunohistochemistry. Protein expressions of PAX7 and IgLON4 were determined by Western blot analysis. Real-time PCR results were normalized to each control (con, before differentiation) and analyzed by t-test. Means ± SD (n ≥3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunocytochemistry, Control, Injection, Muscles, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 2. Inhibitory effect of IgLON4 gene and protein expression on C2C12 myoblast differentiation: (A) mRNA and protein expressions of IgLON4 as determined by real-time RT-PCR and Western blot analysis in Wt and IgLON4kd C2C12 myoblasts on differentiation days (DDs) 2, 4, and 6. mRNA levels on DDs 2, 4 and 6 were normalized versus Wt levels. (B) Expression of MYH by immunocytochemistry in Wt and IgLON4kd C2C12 myoblasts on DD4, and IgLON4kd fusion indices. (C) mRNA and protein levels of MYOD, IgLON5, MYOG, and MYH as determined by real-time RT-PCR and Western blot analysis in Wt and IgLON4kd cells on DDs 2, 4, and 6. mRNA and protein levels were compared at each time point. (D) Morphologies of cells treated with or without IgLON4 antibody on DD2 or 4. mRNA and protein expressions of IgLON4, IgLON5, MYOD, MYOG, and MYH in cells treated with or without IgLON4 antibody on DDs 2 or 4. mRNA and protein levels at each time-point were compared. Wt indicates transfection with scrambled vector. Real-time PCR results were normalized to each control (con, before differentiation) and analyzed by t-test. Means ± SDs (n ≥3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunocytochemistry, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 3. Differentiation, adhesion, and proliferation of Wt, IgLON4kd, IgLON5kd, and Dbkd C2C12 myoblasts: (A) The mRNA and protein expressions of IgLON4 and IgLON5 in Wt and Dbkd C2C12 myoblasts on differentiation day 2 (DD2) as determined by real-time RT-PCR and Western blot. (B) mRNA and protein expressions of MYOD, MYOG, and MYH in Wt and Dbkd cells on DDs 2, 4, or 6 as determined by real-time RT-PCR and Western blot analysis. mRNA and protein levels were compared at each time point. (C) Wt and Dbkd C2C12 myoblast morphologies, MYH expressions (as determined by immunocytochemistry), and fusion indices of Dbkd cells on DD4. (D,E) MTS assays

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Quantitative RT-PCR, Western Blot, Immunocytochemistry

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 4. IgLON4 protein expression and myotube orientation in Wt and IgLON4kd C2C12 myoblasts during differentiation: (A) IgLON4 protein locations were determined by immunocytochemistry on differentiation days (DDs) 2 and 4. Phalloidin and IgLON4 were fluorescently labeled green and red, respectively. Phalloidin was used for counterstaining cytoskeletal actin filaments. (B) Myotube for- mation was observed by MYH immunocytochemistry in Wt and IgLON4kd cells on DD4. Directional analysis of myotube formation by Wt and IgLON4kd cells was performed using ImageJ on DD4. Wt indicates transfection with a scrambled vector.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Expressing, Immunocytochemistry, Labeling, Transfection, Plasmid Preparation

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 5. Locations of IgLON4 protein and lipid rafts on membranes, and the inhibitory effects of IgLON4 mRNA and protein on lipid raft during myoblast differentiation: (A) Locations of IgLON4 protein and lipid rafts as determined by immunocytochemistry on DD2. (B) Locations of IgLON5, NCAM, and CDH15 proteins and lipid rafts as determined by immunocytochemistry on DD2. (C) mRNA and protein expressions of NCAM, CDH15, WASP, CAV1, CAV2, CAV3, and FLOT1 in Wt and IgLON4kd C2C12 myoblasts on DD2 as determined by real-time RT-PCR and Western blot analysis. (D) Extraction of lipid rafts from Wt and IgLON4kd C2C12 myoblasts on DD2, and the protein expressions of IgLON4, IgLON5, CDH15, and FLOT1 by Western blot analysis. FLOT1 and β-actin were used as markers of lipid rafts and total cell lysates, respectively. (E) Locations of IgLON4 protein and lipid rafts in C2C12 myoblasts treated with or without IgLON4 antibody as determined by immunocytochemistry on DD2. Wt indicates transfection with a scrambled vector. Lipid rafts were labeled green using cholera toxin and IgLON4, IgLON5, NCAM, and CDH15 were labeled red using Alexa Fluor 594. Real-time PCR results were normalized to each control (con, before differentiation) and analyzed by t-test. Means ± SDs (n ≥3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Immunocytochemistry, Quantitative RT-PCR, Western Blot, Extraction, Transfection, Plasmid Preparation, Labeling, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 6. Effect of using striped culture plates on C2C12 myoblasts differentiation: (A) The formation of myotubes in normal and striped plate cultures was observed by MYH immunocytochemistry. (B) mRNA and protein expressions of myogenic markers, muscle-specific adhesion proteins, and IgLON family members in C2C12 myoblasts cultured in normal and striped plates on DD2 and 4 as determined by real-time RT-PCR and Western blot analysis. (C) Location of IgLON4 protein as determined by immunocytochemistry on DD2 in C2C12 myoblasts cultured on striped plates. Labeling was performed using fluorescently labeled phalloidin and IgLON4 (green and red, respec- tively). Phalloidin was used to counterstain cytoskeletal actin filaments. (D) Locations of lipid rafts and IgLON4 protein as determined by immunocytochemistry on DD2. Lipid rafts were stained using labeled cholera toxin (green fluorescence) and IgLON4 with Alexa Fluor 594 (red fluorescence). (E) Morphologies of C2C12 myoblasts treated with or without IgLON4 antibody on DD2. Real-time PCR results were normalized to each control (con, before differentiation) and analyzed by t-test. Means ± SD (n ≥3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Immunocytochemistry, Cell Culture, Quantitative RT-PCR, Western Blot, Labeling, Staining, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 7. The proposed role of IgLON4 during myoblast differentiation. (A) Schematic structure of IgLON4, its membrane location, and its association with lipid raft accumulation during my- oblast differentiation (B) Illustrations of the hypothetical structure of IgLON4-blocked muscle and normal muscle.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Membrane