Journal: Pharmaceutics

Article Title: Mesenchymal Stem/Stromal Cells in Skeletal Muscle Are Pro-Angiogenic, and the Effect Is Potentiated by Erythropoietin

doi: 10.3390/pharmaceutics15041049

Figure Lengend Snippet: Production of angiogenic cytokines in mMSCs. ( a ) Representative microscopic images of the mMSCs. Left phase contrast image. Original magnification ×40. Right, red fluorescence indicates PDGFR-α. PDGFR-α was ubiquitously expressed in the mMSCs. Nuclei were stained with DAPI (blue). Bar = 50 µm. ( b ) Concentration levels of VEGF and HGF in the conditioned medium. N = 6. * p < 0.05 vs. BMMSCs. # p < 0.01 vs. skMCs. mMSCs, mesenchymal stem/stromal cells derived from skeletal muscle; PDGFR-α, platelet-derived growth factor receptor-α; DAPI, 4’,6-Diamidino-2-phenylindole; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor; BMMSCs, bone marrow mesenchymal stem/stromal cells; skMCs, skeletal muscle cells.

Article Snippet: Rat skeletal muscle cells (skMCs) and human umbilical cord vein endothelial cells (HUVECs) were purchased from Cell Applications Inc. (San Diego, CA, USA).

Techniques: Fluorescence, Staining, Concentration Assay, Derivative Assay

Journal: Pharmaceutics

Article Title: Mesenchymal Stem/Stromal Cells in Skeletal Muscle Are Pro-Angiogenic, and the Effect Is Potentiated by Erythropoietin

doi: 10.3390/pharmaceutics15041049

Figure Lengend Snippet: Proliferation of cultured mMSCs by Erythropoietin (Epo). ( a ) Left, Epo-R (green) expression in mMSCs. Nuclei were stained with DAPI (blue). NC, negative control. Bar = 50 µm. Right, western blot analysis. Epo stimulated the phosphorylation of Akt and STAT3 in the mMSCs. Epo-R, erythropoietin receptor. ( b ) Efficacy of Epo stimulation on the propagation of HUVECs, skMCs, and mMSCs. N = 3 to 6. # p < 0.01. HUVECs, human umbilical cord vein endothelial cells.

Article Snippet: Rat skeletal muscle cells (skMCs) and human umbilical cord vein endothelial cells (HUVECs) were purchased from Cell Applications Inc. (San Diego, CA, USA).

Techniques: Cell Culture, Expressing, Staining, Negative Control, Western Blot, Phospho-proteomics

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

doi: 10.1152/ajpendo.00238.2010

Figure Lengend Snippet: Effects of free fatty acids (FFAs) and ceramide on mitochondrial reactive oxygen species (mtROS) production in L6 myotubes. After 24 h of treatment, cells were analyzed in a fluorescent plate reader, and the increase in ROS production was calculated as a %increase compared with control. Mitochondrial superoxide production in L6 myotubes treated with the indicated concentrations of the different FFAs (A), the indicated concentrations of C2-ceramide (C2) or metabolically inactive C2-dihydroceramide (C2-di) (B), or the indicated concentrations of palmitate in the presence of 50 μM fumonisin B1 (FB1; C). The mean results ± SE are shown (n ≥ 3). *P < 0.05. o/p mix, Oleate-palmitate mixture.

Article Snippet: Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA).

Techniques: Control, Metabolic Labelling

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

doi: 10.1152/ajpendo.00238.2010

Figure Lengend Snippet: Oleate protects L6 myotubes against palmitate-induced mitochondrial DNA (mtDNA) damage and decline in ATP level. A: break frequency per 10.8 kb fragment of mtDNA after 6 h of treatment with the indicated concentrations of palmitate, oleate, or the o/p mixture. Break frequency was determined as described in materials and methods. Intensity of the band was determined by densitometry. B: L6 myotubes were treated with the indicated concentration of palmitate, oleate, or o/p mix for 24 h, and ATP production was measured. The mean results ± SE are shown (n ≥ 3). *P < 0.05.

Article Snippet: Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA).

Techniques: Concentration Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

doi: 10.1152/ajpendo.00238.2010

Figure Lengend Snippet: Cell viability in L6 myotubes after treatment with FFAs and ceramide. A: only palmitate significantly decreased viability in L6 myotubes. B: C2-ceramide but not C2-di diminished mitochondrial viability in L6 myotubes. C: FB1 blocked de novo synthesis of ceramide and enhanced viability in L6 myotubes. The mean results ± SE are shown (n ≥ 3). *P < 0.05.

Article Snippet: Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA).

Techniques:

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

doi: 10.1152/ajpendo.00238.2010

Figure Lengend Snippet: Effects of palmitate, oleate, and the o/p mixture on JNK activation in skeletal muscle cells. L6 myotubes were exposed to the indicated concentrations of palmitate, oleate, or o/p mix for 24 h. Total cell lysates were isolated and analyzed by Western blot with the indicated antibodies. A: representative blots are shown. B: the values from densitometry from at least 3 (p-JNK) independent experiments were normalized to the level of total JNK and expressed as fold of difference compared with the corresponding untreated controls ± SE (n ≥ 3). *P < 0.001.

Article Snippet: Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA).

Techniques: Activation Assay, Isolation, Western Blot

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

doi: 10.1152/ajpendo.00238.2010

Figure Lengend Snippet: Oleate prevents palmitate-induced apoptosis in skeletal muscle cells. L6 myotubes were treated with the indicated concentrations of palmitate, oleate, or o/p mix for 24 h. Caspase-3 antibodies were used to recognize the full-length (35 kDa) and cleaved (17 kDa) fragments of caspase-3. Representative blots from 3 independent experiments are shown. Equal loading was confirmed using anti-actin antibody.

Article Snippet: Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA).

Techniques:

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

doi: 10.1152/ajpendo.00238.2010

Figure Lengend Snippet: Oleate, N-acetylcysteine (NAC), and FB1 ameliorated palmitate-mediated inhibition of insulin-induced Akt (Ser473) phosphorylation in skeletal muscle cells. A and B: L6 myotubes were exposed to the indicated concentrations of palmitate, oleate, or o/p mix for 16 h and then serum starved for 2 h and incubated in the presence or absence of 100 nM insulin for 15 min. Total cell lysates were isolated and analyzed by Western blot analysis with the indicated antibodies. A: representative blots from at least 3 independent experiments are shown. B: the values from densitometry from at least 3 (p-Akt) independent experiments were normalized to the level of total Akt and expressed as fold of difference after addition of insulin compared with the corresponding untreated controls ± SE (n ≥ 3). *P < 0.001. C: L6 myotubes were incubated with 1 mM palmitate (P) alone or with 1 mM palmitate in the presence of either 5 mM NAC or 50 μM FB1. Western blot analysis was performed using p-Akt and total Akt antibodies. C, control.

Article Snippet: Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA).

Techniques: Inhibition, Phospho-proteomics, Incubation, Isolation, Western Blot, Control

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

doi: 10.1152/ajpendo.00238.2010

Figure Lengend Snippet: Effect of a JNK inhibitor on palmitate-induced inhibition of insulin-stimulated Akt (Ser473) phosphorylation and apoptosis in L6 myotubes. To evaluate insulin signaling, L6 myotubes were incubated with 1 mM palmitate in the presence or absence of 25 μM JNK inhibitor SP-600125 (SP) for 16 h prior to serum starvation and stimulation with insulin. To assess apoptosis, L6 myotubes were incubated with 1 mM palmitate with or without 25 μM JNK inhibitor SP for 24 h. Representative blots are shown. Western blots analyses were performed using p-Akt and total Akt antibodies (A) and caspase-3 and actin antibodies (B).

Article Snippet: Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA).

Techniques: Inhibition, Phospho-proteomics, Incubation, Western Blot

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

doi: 10.1152/ajpendo.00238.2010

Figure Lengend Snippet: Effects of palmitate, oleate, or o/p mix on the expression of mitochondrial transcription factor (TFAM) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and on the promoter activity of PGC-1α in skeletal muscle cells. L6 myotubes were exposed to the indicated concentrations of palmitate, oleate, or o/p mix for 24 h. A: total cell lysates were isolated and analyzed by Western blot with the indicated antibodies. Representative blots are shown. B: the values from densitometry from at least 3 independent Western blot experiments for PGC-1α were normalized to the level of actin and presented as fold of difference compared with the corresponding untreated controls ± SE (n ≥ 3). #P < 0.01 vs. o/p mix; *P < 0.05 vs. oleate. C: densitometry results for TFAM protein normalized for actin level and presented as fold of difference compared with the corresponding untreated controls ± SE (n ≥ 3); *P < 0.05 vs. palmitate. D: L6 myotubes were transiently transfected with a mixture of PGC-1α promoter and pEGFP-N1 plasmids. Cells were treated with the indicated concentrations of palmitate, 2-bromopalmitate, oleate, or o/p mix, and the PGC-1α promoter activity was determined as described in materials and methods. Luciferase expression was normalized to green fluorescent protein (GFP) fluorescence intensity. Data are indicated as a percentage of the untreated controls. Means ± SE are shown (n ≥ 3). *P < 0.01 vs. both oleate and o/p mix.

Article Snippet: Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA).

Techniques: Expressing, Activity Assay, Isolation, Western Blot, Transfection, Luciferase, Fluorescence

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

doi: 10.1152/ajpendo.00238.2010

Figure Lengend Snippet: ROS scavenger reversed palmitate-induced downregulation of both TFAM and PGC-1α protein level and PGC-1α promoter activity. L6 myotubes were pretreated in the presence or absence of 5 mM NAC for 30 min and then exposed to the indicated concentrations of palmitate. NAC significantly increased the palmitate-induced decline in TFAM (A) and PGC-1α expression (B). C: L6 myotubes were transiently transfected with a mixture of PGC-1α promoter and pEGFP-N1 plasmids. Cells were preincubated in the presence or absence of 5 mM NAC for 30 min and then exposed to the indicated concentrations of palmitate, and PGC-1α promoter activity was determined. Luciferase expression was normalized to GFP fluorescence intensity. Data are indicated as a percentage of untreated controls. Means ± SE are shown (n ≥ 3). *P < 0.01 vs. palmitate.

Article Snippet: Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA).

Techniques: Activity Assay, Expressing, Transfection, Luciferase, Fluorescence