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: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are localized and secreted by human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A Human lung tissue from control subjects, idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF was immune-stained with IL-11, IL-11Rα and αSMA and with secondary fluorescence antibodies. Representative images are showed. White colour represents co-localization of both antibodies. Yellow arrows indicate endothelial cells. B HPAECs and C HPASMCs were isolated from pulmonary arteries of control subjects, IPF and PH associated to IPF patients and cultured until passage 1. Cell culture supernatants were collected to measure IL-11 by ELISA. Data are presented as scatter dot blot with median and interquartile range values of n = 6 patients in each group. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Staining, Fluorescence, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are increased in whole lung homogenates, isolated pulmonary arteries and serum of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF. The protein expression of IL-11 and IL-11Rα in A , B isolated pulmonary arteries (70–500 µm of internal diameter), C , D serum, and E , F lung tissue homogenates. Protein expression was measured using ELISA kits. H CD31 protein expression was measured in isolated pulmonary arteries as endothelial cells marker by ELISA. H , I Human lung tissue from control subjects, IPF and PH associated to IPF was immune-stained with IL-11, IL-11Rα and alpha smooth muscle actin (αSMA) antibodies. Representative images are showed from non-fibrotic lung areas and fibrotic areas. J Vascular wall thickening was quantified in a total of 20–30 pulmonary arteries per patient. K , L Immunohistochemical score quantification of IL-11 and IL-11Rα in a total of 20–30 pulmonary arteries per patient. Scale bar: 100 µm. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison. M Spearman ρ correlation of IL-11 expression in isolated pulmonary arteries from PH + IPF and mean pulmonary artery pressure (mPAP). N indicates the number of patients in each graph

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Marker, Staining, Immunohistochemical staining, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: SiRNA-IL-11 transiently transfection attenuates bleomycin-induced lung fibrosis and pulmonary hypertension in transgenic Tie2-GFP mice. Wild-type (WT) siRNA(−) Tie2-GFP mice and IL-11-KO siRNA-IL-11 Tie2-GFP mice received a single intratracheal dose of bleomycin (1.5 U/kg) on day 1 ( n = 11) during 14 days. siRNA-IL-11 was administered intravenously and intranasally three times a week from day 1 to day 14. At day 14 the following parameters were measured. A Masson’s trichrome histological images are showed. Scale bar: 100 µm. B Ashcroft score lung fibrotic index, C hydroxyproline amount in lung tissue D right ventricular systolic pressure (RVSP) mmHg, E right ventricular (RV) hypertrophy measured by the ratio of RV/left ventricular (LV) + septo in mg/mg, F pulmonary artery remodeling and G inflammatory cells in bronchoalveolar lavage fluid (BALF) were measured. H Immunohistochemical analysis of αSMA, IL-11 and IL-11Rα. Scale bar: 50 µm. Black arrows show pulmonary arteries. I Co-immunofluorescence of αSMA/Tie2-GFP. Scale bar: 25 µm. White arrows indicates co-localizations. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Transfection, Transgenic Assay, Immunohistochemical staining, Immunofluorescence, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and soluble IL-11Rα induce human pulmonary artery endothelial cell (HPAEC) to mesenchymal transition (EnMT) and human pulmonary artery smooth muscle cell (HPASMC) to myofibroblast-like transition. A HPAEC and B HPASMC were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or their combination during 48 h replacing culture medium and stimulus each 24 h. Experiments were done between passages 2–3. Gene mRNA transcripts of different genes measured by quantitative PCR (qPCR) as 2 −ΔCt . Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin for protein. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 4 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα activates intracellular signal. A Human pulmonary artery endothelial cells (HPAEC) and B human pulmonary artery smooth muscle cells (HPASMC) were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination during 30 min. Experiments were done between passages 2–3. Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin or non-phosphorylated protein as indicate. Representative blots are sowed. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα promotes time-dependent proliferation and senescence in human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A HPAECs or B HPASMCs were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination at indicated times. Experiments were done between passages 2–3. Cell proliferation was measured by the BrDU kit at 24 h, 48 h, 72 h and 96 h. Cell senescence was measured after 72 h of cell stimulation using β-galactosidase histology and P21 expression. Results were expressed as % senescence (β-galactosidase blue positive cells) relative to the total number of cells in each field. P21 expression was measured by quantitative PCR (qPCR) as 2 −ΔCt and western blot. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3–4 control subjects performed in triplicate). P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Cell Stimulation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Dot Blot

Journal: Scientific Reports

Article Title: Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF 164 in skeletal muscle

doi: 10.1038/srep21546

Figure Lengend Snippet: In vivo expression of the Vegfa and Pdgfb genes, discriminating between the delivered and endogenous transcripts, was quantified in calf muscles 4 and 7 days after implantation with control cells (Ctrl) and myoblast clones expressing medium or high levels of VEGF alone or with PDGF-BB (V and VIP, respectively). Data are shown as mean ± SEM (n = 4 muscles/condition); *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 (ANOVA with Sidak multiple comparisons test after data normalization by logarithmic transformation).

Article Snippet: To determine the expression of the other genes the following TaqMan gene expression assays were used from Applied Biosystems (Applied Biosystems, Zug, Switzerland): murine Pdgfb (Mm00440678_s1), human Pdgfb (Hs00234042_m1) and murine Gapdh housekeeping gene (Mm03302249_g1).

Techniques: In Vivo, Expressing, Muscles, Control, Clone Assay, Transformation Assay