Journal: International Journal of Molecular Sciences

Article Title: All- Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion

doi: 10.3390/ijms24109035

Figure Lengend Snippet: Expression pattern and role of LGR6 during myoblast differentiation. ( A ) C2C12 myoblasts were differentiated into myotubes for 5 days. The expression level of Lgr6 mRNA was determined by qPCR. ( B ) Myoblasts were differentiated into myotubes for 24 h. The levels of mRNAs encoding LGR6 and myogenic regulatory factors were determined by qPCR. ( C ) Myoblasts were transfected with control siRNA (siControl) or Lgr6 siRNA (siLGR6 #1 and siLGR6 #2), and cells were harvested 0 h after the induction of differentiation. The Lgr6 mRNA levels were determined by qPCR. ( D ) Myoblasts were differentiated for 3 days after siRNA transfection. Fixed cells were fluorescently labeled using an anti-MyHC antibody and fluorescence-labeled secondary antibody (green). The nuclei were stained with DAPI (blue). Bars, 100 μm. ( E ) The differentiation and fusion indices were calculated. The percentage of MyHC-positive cells with one, two, or three more nuclei was determined. ( F ) Myoblasts were differentiated for 24 h after siRNA transfection, and cells were harvested. The levels of mRNAs for myogenic regulatory factors were determined by qPCR. ( G ) Myoblasts were differentiated into myotubes. LGR6 expression was analyzed by Western blotting. Arrow indicates the lower band. ( H ) LGR6 levels were normalized to β-actin levels. ( A , B , H ) The results are presented as the mean ± SD ( n = 3). Data were determined using one-way ANOVA and Tukey’s post hoc test. ( A , H ) Columns with different letters are significantly different at p < 0.05, whereas columns sharing the same letters are not significantly different. ( B ) Different letters with the same color on the lines indicate statistically significant differences ( p < 0.05). ( C , E , F ) The results are presented as the mean ± SD ( n = 3). Data were determined using one-way ANOVA and Dunnet’s post hoc test. * p < 0.05 vs. siControl.

Article Snippet: Murine C2C12 myoblasts (European Collection of Authenticated Cell Cultures, Salisbury, UK) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (growth medium) and differentiated into myotubes in Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum and antibiotics (differentiation medium), as described previously [ ].

Techniques: Expressing, Transfection, Control, Labeling, Fluorescence, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: All- Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion

doi: 10.3390/ijms24109035

Figure Lengend Snippet: LGR6 expression during myoblast differentiation as an ATRA-responsive gene. ( A ) C2C12 myoblasts were differentiated in the presence or absence of ATRA. The Lgr6 mRNA levels were determined by qPCR. ( B ) Myoblasts were cultured with ATRA in the presence of AGN193109. The Lgr6 mRNA levels were determined by qPCR. ( C ) C2C12 myoblasts were cultured with ATRA, AM580, BMS961, AM580, and BMS961. The Lgr6 mRNA levels were determined by qPCR. ( D ) Myoblasts were cultured with ATRA in the presence or absence of Ro41-5253 and/or LY2955303. The Lgr6 mRNA levels were determined by qPCR. ( E ) Myoblasts were transfected with control siRNA (siControl) or Lgr6 siRNA (siLGR6#1), and cells were harvested immediately after the induction of differentiation. The Lgr6 mRNA levels were determined by qPCR. ( F ) After siRNA transfection, myoblasts were differentiated in the presence or absence of ATRA for 3 days. Fixed cells were immunofluorescently labeled using an anti-MyHC antibody (green), and the nuclei were stained with DAPI (blue). Bars, 100 μm. ( G ) The differentiation and fusion indices were calculated. ( A – D , G ) The results are presented as the mean ± SD ( n = 3). Data were determined using two-way ANOVA and Tukey’s post hoc test. Columns with different letters are significantly different at p < 0.05, whereas columns sharing the same letters are not significantly different. ( E ) The results are presented as the mean ± SD ( n = 3). Data were determined using Student’s t -test. * p < 0.05 vs. siControl.

Article Snippet: Murine C2C12 myoblasts (European Collection of Authenticated Cell Cultures, Salisbury, UK) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (growth medium) and differentiated into myotubes in Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum and antibiotics (differentiation medium), as described previously [ ].

Techniques: Expressing, Cell Culture, Transfection, Control, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: All- Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion

doi: 10.3390/ijms24109035

Figure Lengend Snippet: Involvement of the ubiquitin–proteasome system in LGR6 expression. ( A ) C2C12 myoblasts were transfected with a mock vector and murine and human LGR6 expression vectors, followed by culture in the presence or absence of MG132. LGR6 levels were analyzed by Western blotting. ( B ) Myoblasts were transfected with a mock vector and a human LGR6 expression vector, followed by further culture in the presence or absence of MG132. The LGR6 levels were analyzed by Western blotting. Myc- and His-tagged LGR6 (LGR6-Myc/His) was pulled down using Ni-Sepharose resin, and Ni-Sepharose-bound proteins were analyzed by Western blotting using anti-Myc and anti-ubiquitin antibodies. ( C ) Myoblasts were transfected with a human LGR6 expression vector, followed by further transfection with control siRNA or Znrf3 siRNAs (siZNRF3#1 and siZNRF3#2). As a negative control for exogenous LGR6, myoblasts were transfected with a mock vector. The Lgr6 mRNA levels were determined by qPCR. Data were determined by Student’s t -test. * p < 0.05 vs. siControl. ( D ) Exogenous LGR6 levels were analyzed by Western blotting and normalized to β-actin levels. The results are presented as the mean ± SD ( n = 3). Data were determined using one-way ANOVA and Dunnet’s post hoc test. * p < 0.05 vs. siControl. ( E ) Deduced positions of lysine residues (yellow stars: K598, K679, and K861) in the intracellular region of human LGR6. ( F ) Myoblasts were transfected with a mock vector and wild-type or mutant-type human LGR6 expression vectors, followed by further culture in the presence or absence of MG132. LGR6 expression was analyzed by Western blotting using an anti-Myc antibody.

Article Snippet: Murine C2C12 myoblasts (European Collection of Authenticated Cell Cultures, Salisbury, UK) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (growth medium) and differentiated into myotubes in Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum and antibiotics (differentiation medium), as described previously [ ].

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Negative Control, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: All- Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion

doi: 10.3390/ijms24109035

Figure Lengend Snippet: Involvement of LGR6 in Wnt/β-catenin signaling. ( A ) C2C12 myoblasts were transfected with a TCF reporter expression vector, followed by further incubation in the presence or absence of RSPO2. TCF activity was determined. ( B ) Myoblasts were transfected with a TCF reporter expression vector and a Wnt3a expression vector, followed by further incubation in the presence or absence of RSPO2. TCF activity was determined. ( C ) Myoblasts were transfected with a TCF reporter expression vector and a Wnt3a expression vector and further transfected with control siRNA (siControl) or Lgr6 siRNAs (siLGR6 #1 and siLGR6 #2). Cells were harvested immediately after the induction of differentiation. The Lgr6 mRNA levels were determined by qPCR. ( D ) After siRNA transfection, myoblast differentiation was induced in the presence or absence of RSPO2. TCF activity was determined. ( A , B , D ) The results are presented as the mean ± SD ( n = 3). Data were determined using two-way ANOVA and Tukey’s post hoc test. Columns with different letters are significantly different at p < 0.05, whereas columns sharing the same letters are not significantly different. ( C ) The results are presented as the mean ± SD ( n = 3). Data were determined using Student’s t -test. * p < 0.05 vs. siControl.

Article Snippet: Murine C2C12 myoblasts (European Collection of Authenticated Cell Cultures, Salisbury, UK) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (growth medium) and differentiated into myotubes in Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum and antibiotics (differentiation medium), as described previously [ ].

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Activity Assay, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Distribution of estrogen receptors in various organs and tissues of adult mouse. a Western blot analysis of whole-cell fractions with a specific monoclonal estrogen receptor (ER) antibody. Upper panel immunoreactive bands corresponding to the ER protein (66 kDa). The histogram (lower) indicates the results of densitometric analysis for a 66-kDa band (mean ± SE, n = 4). Relative expression level with respect to the ovary is shown in %. From left to right: skeletal muscle, uterus, lung, adipose tissue, kidney, and myocardium. b Relative expression (to ovary) of whole cell and nonnuclear ER proteins obtained from C2C12 mouse myoblasts (n = 3)

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Western Blot, Expressing

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Time course of ER mRNA and protein expression during E2 exposure. a Representative of C2C12 mRNA transcripts for ER and GAPDH at several time points after exposure to E2 (10−8 M) (upper). The mRNA level of ER relative to GAPDH is averaged from five individual experiments and displayed as mean ± SE (statistically significant). At 0, 0.5, 1, 3, and 5 h (lower). b Proteins were extracted from nucleus-free C2C12 cells exposed to E2 (10−8 M) for varying periods of time (from left: 0, 1, 3, 4, 6, 12, and 24 h) and immunoblotted with the ER antibody. Immunodensity in a 66-kDa band is quantified by densitometry and presented as the % increase of control (time 0). Symbols and bars represent mean ± SE (n = 5); statistically significant

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Expressing, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Dose–dependent effects of 17β-estradiol on the expression of total and nonnuclear ER. Representative immunoreactive bands for total ER in C2C12 myoblasts (upper panel) and the relative expression of total ER protein (a) or nonnuclear ER protein (b) quantified by densitometric analysis (lower panel); after treatment with either vehicle (control), different concentrations of 17β-estradiol (10−12–10−5 M) in a and (10−10–10−6 M) in b for 15 h. c Shows the relative expression of total ER protein after treatment with vehicle (control), 17β-estradiol (10−8 M), and BSA-conjugated E2 (10−8 M) for 15 h. Each bar is normalized to the control (far left) and expressed as %. Columns and bars indicate mean ± SE (n = 3 in a, n = 4 in b, and n = 5 in c). *P < 0.01, ☆ P < 0.05 compared with control values, determined using Student’s t test

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Expressing, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Influence of 17-βestradiol on C2C12 proliferation. C2C12 cells were grown in the serum-free medium for 48 h, which was then changed to ones with or without E2 (10−8 M) in 1% FBS (open triangle, closed triangle) or 10% FBS (open circle, closed circle). The rate of cell growth is shown as the cell number at every 24 h relative to 0 h. Symbols and bars represent the mean ± SE (n = 5)

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques:

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Effects of ER antagonists on E2-induced whole-cell and non-nuclear ER increase. C2C12 cells were treated with E2 (10−8 M) in the absence or presence of either tamoxifen or ICI 182,780 (10−7 M; a total ER) or (10−5 M; b non-nuclear fractions) for 15 h. Tamoxifen or ICI 182,780 was added to the culture medium 30 min prior to the administration of E2. Total and nonnuclear fractions of ER were prepared as described in “Materials and methods” and subjected to Western blotting. Histograms represent the extent of inhibition of E2-induced ER increase in total a and nonnuclear b fractions, which are expressed as the percentage of control (no stimulation), averaged from five separate experiments (mean ± SE). *P < 0.01, compared with control values, determined using Student’s t test

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Western Blot, Inhibition, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Effects of 17β-estradiol and/or TPA on ER de novo synthesis. In the absence (control) or presence of 17β-estradiol (10−8 M) and/or TPA (10−6 M), C2C12 cells were labeled with [35S] methionine. At the time points indicated in the figure, the total ER fractions were prepared and subjected to selective immunoprecipitation for ER. The immunoprecipitates were analyzed by SDS-PAGE and image scanning. Data are the mean ± SE from three separate experiments. Left panel time course of 35methionine incorporation in ER. Right panel rates of the increase at 5 h by the relative ratio of control. *P < 0.01, compared with control values, determined using Student’s t test. ☆ P < 0.01, compared with E2 values, determined using Student’s t test

Article Snippet: C2C12 murine skeletal muscle cell line [ 20 ], obtained from Dainippon Pharmaceutical Ltd., was routinely cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) containing 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS).

Techniques: Control, Labeling, Immunoprecipitation, SDS Page

Journal: Cells

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

doi: 10.3390/cells11203265

Figure Lengend Snippet: 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 (differentiation 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 regeneration (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: Murine C2C12 myoblast cells (Korean Cell Line Bank, Seoul, Korea) were cultured in growth media DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS (Fetal Bovine Serum) and 1% P/S (penicillin/streptomycin; all were purchased from Hyclone, South Logan, UT, USA) in a humidified 5% CO 2 incubator at 37 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunocytochemistry, Control, Injection, Muscles, Immunofluorescence, 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: 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 IgLON4 kd 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 IgLON4 kd C2C12 myoblasts on DD4, and IgLON4 kd 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 IgLON4 kd 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: Murine C2C12 myoblast cells (Korean Cell Line Bank, Seoul, Korea) were cultured in growth media DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS (Fetal Bovine Serum) and 1% P/S (penicillin/streptomycin; all were purchased from Hyclone, South Logan, UT, USA) in a humidified 5% CO 2 incubator at 37 °C.

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: Differentiation, adhesion, and proliferation of Wt, IgLON4 kd , IgLON5 kd , and Db kd C2C12 myoblasts: ( A ) The mRNA and protein expressions of IgLON4 and IgLON5 in Wt and Db kd 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 Db kd 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 Db kd C2C12 myoblast morphologies, MYH expressions (as determined by immunocytochemistry), and fusion indices of Db kd cells on DD4. ( D , E ) MTS assays were used to assess Wt, IgLON4 kd , IgLON5 kd , and Db kd C2C12 myoblast cell adhesions and proliferation after differentiation for 3 h or 3 days, respectively. ( F ) Wt, IgLON4 kd , IgLON5 kd , and Db kd cells were cultured until 30% or 100% confluent, or for 4 days. Plates were then sealed and placed inverted and centrifuged using a swinging bucket rotor at 100×, 500×, or 1000× g . Relative cell counts after centrifugation, as assessed by MTS assay and normalized versus initial cell numbers (before centrifugation). Wt indicates transfection with a 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: Murine C2C12 myoblast cells (Korean Cell Line Bank, Seoul, Korea) were cultured in growth media DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS (Fetal Bovine Serum) and 1% P/S (penicillin/streptomycin; all were purchased from Hyclone, South Logan, UT, USA) in a humidified 5% CO 2 incubator at 37 °C.

Techniques: Quantitative RT-PCR, Western Blot, Immunocytochemistry, Cell Culture, Centrifugation, MTS Assay, 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: IgLON4 protein expression and myotube orientation in Wt and IgLON4 kd 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 formation was observed by MYH immunocytochemistry in Wt and IgLON4 kd cells on DD4. Directional analysis of myotube formation by Wt and IgLON4 kd cells was performed using ImageJ on DD4. Wt indicates transfection with a scrambled vector.

Article Snippet: Murine C2C12 myoblast cells (Korean Cell Line Bank, Seoul, Korea) were cultured in growth media DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS (Fetal Bovine Serum) and 1% P/S (penicillin/streptomycin; all were purchased from Hyclone, South Logan, UT, USA) in a humidified 5% CO 2 incubator at 37 °C.

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: 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 IgLON4 kd C2C12 myoblasts on DD2 as determined by real-time RT-PCR and Western blot analysis. ( D ) Extraction of lipid rafts from Wt and IgLON4 kd 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: Murine C2C12 myoblast cells (Korean Cell Line Bank, Seoul, Korea) were cultured in growth media DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS (Fetal Bovine Serum) and 1% P/S (penicillin/streptomycin; all were purchased from Hyclone, South Logan, UT, USA) in a humidified 5% CO 2 incubator at 37 °C.

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: 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, respectively). 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: Murine C2C12 myoblast cells (Korean Cell Line Bank, Seoul, Korea) were cultured in growth media DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS (Fetal Bovine Serum) and 1% P/S (penicillin/streptomycin; all were purchased from Hyclone, South Logan, UT, USA) in a humidified 5% CO 2 incubator at 37 °C.

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