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

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: Biochemical Journal

Article Title: GSK3-mediated raptor phosphorylation supports amino-acid-dependent mTORC1-directed signalling

doi: 10.1042/BJ20150404

Figure Lengend Snippet: ( A – C ) Cells were held in EBSS containing or lacking AAs for 1 h or alternatively having been depleted of AAs incubated in EBSS containing a 1× AA mix (refeed) for 15 min. Cells were incubated with 100 nM rapamycin, 50 μM SB415286 or 30 μM roscovitine for 15 min prior to and for 15 min during the AA-refeed period. Cells were lysed and 30 μg of lysate protein was analysed by immunoblotting. Blots shown in ( A – C ) are representative of a minimum of three independent experiments and histograms (means ± S.E.M., * P <0.05) show quantification of these. ( D ) L6 myotubes were AA-depleted for 1 h in EBSS followed by incubation in EBSS+AA for 15 min in the absence or presence of 50 μM SB415286, 10 μM SB216763, 30 μM roscovitine, 40 μM CT99021 or 100 nM rapamycin for 0.5 h. Cells were harvested and 30 μg of protein was analysed by immunoblotting using the antibodies indicated. ( E ) HEK293T cells were subjected to AA-depletion/refeeding as in ( A – C ) or incubated with EBSS supplemented with serum [10% (v/v) FBS] ± 100 nM rapamycin or 50 μM SB415286 for 0.5 h, as indicated. Cells were lysed and 30 μg of protein was analysed by immunoblotting using the antibodies indicated.

Article Snippet: L6 rat skeletal muscle cells were grown as described previously in α-MEM containing 2% (v/v) FBS (Biosera) [ ].

Techniques: Incubation, Western Blot

Journal: Biochemical Journal

Article Title: GSK3-mediated raptor phosphorylation supports amino-acid-dependent mTORC1-directed signalling

doi: 10.1042/BJ20150404

Figure Lengend Snippet: ( A ) Total RNA was extracted from L6 myotubes stably expressing either a non-specific shRNA or shRNAs targeting GSK3α and GSK3β. cDNA was synthesized from the mRNA and relative GSK3 mRNA abundance, as measured against GAPDH mRNA, assessed by quantitative PCR. ( B ) Lysates were prepared from L6 myotubes stably expressing either a non-specific shRNA or shRNAs targeting GSK3α and GSK3β. A 30 μg amount of lysate was analysed by immunoblotting using anti-GSK3α/β or anti-GAPDH antibodies. In addition, 100 μg of lysate was also subjected to immunoprecipitation using antibodies against either GSK3α or GSK3β and analysis of the respective GSK3 activities carried out using a phospho-GS peptide as a substrate. ( C ) L6 myotubes stably expressing either a non-specific shRNA or shRNAs targeting GSK3α and GSK3β were held in EBSS containing or lacking AA for 1 h or alternatively having been depleted of AAs incubated in EBSS containing a 1× physiological AA mix (refeed) for 15 min in the absence or presence of 50 μM SB415286. Cells were harvested and 30 μg of lysate was analysed by immunoblotting using the antibodies indicated. The asterisk indicates a significant ( P <0.05) change between the indicated bars.

Article Snippet: L6 rat skeletal muscle cells were grown as described previously in α-MEM containing 2% (v/v) FBS (Biosera) [ ].

Techniques: Stable Transfection, Expressing, shRNA, Synthesized, Real-time Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Incubation

Journal: Biochemical Journal

Article Title: GSK3-mediated raptor phosphorylation supports amino-acid-dependent mTORC1-directed signalling

doi: 10.1042/BJ20150404

Figure Lengend Snippet: ( A ) HeLa cells were cultured on coverslips and transfected with GFP-tagged TFEB. Twenty-four hours post transfection, cells were incubated in EBSS and EBSS+AA in the absence or presence of 100 nM rapamycin or 50 μM SB415286 as indicated and cells were fixed, imaged and analysed as described in the Materials and methods section. Asterisks indicate significant ( P <0.05) changes between indicated bars. ( B ) HeLa cells, L6 myotubes and U2OS cells were subjected to incubation with EBSS containing or lacking AAs for 1 h or alternatively having been depleted of AAs incubated in EBSS containing a 1× AA mix (refeed) for 15 min in the absence/presence of 100 nM rapamycin, 50 μM SB415286 or 30 μM roscovitine as indicated. Cells were lysed and 30 μg of lysate protein was analysed by immunoblotting. ( C ) U20S cells were incubated as in ( B ) but in the absence or presence of 50 nM bafilomycin A1. Cells were fixed and nuclei (blue DAPI) and LC3 puncta (green fluorescence) visualized as described in the Materials and methods section. Asterisks indicate a significant difference between the indicated bars ( A ) or indicate a significant difference in LC3 puncta observed relative to the untreated AA-sufficient control ( P <0.05).

Article Snippet: L6 rat skeletal muscle cells were grown as described previously in α-MEM containing 2% (v/v) FBS (Biosera) [ ].

Techniques: Cell Culture, Transfection, Incubation, Western Blot, Fluorescence, Control

Journal: Biochemical Journal

Article Title: GSK3-mediated raptor phosphorylation supports amino-acid-dependent mTORC1-directed signalling

doi: 10.1042/BJ20150404

Figure Lengend Snippet: ( A and B ) L6 myotubes were either held in EBSS containing or lacking AAs for 1 h or alternatively, having been AA-depleted, incubated in EBSS containing a 1× AA mix (refeed) for 15 min. Cells were incubated with 100 nM rapamycin, 50 μM SB415286 or 30 μM roscovitine for 15 min prior to and for 15 min during the AA-refeed period as indicated prior to cell lysis. Alternatively, L6 myotubes stably expressing non-specific shRNA or shRNAs targeting GSK3α/GSK3β were lysed for analysis. A 30 μg amount of lysate was analysed by immunoblotting using the antibodies indicated. ( C ) Effect of GSK3 inhibition on raptor and mLST8 association with mTOR and 4E-BP1 phosphorylation. HEK293T cells were incubated in EBSS lacking or containing AAs and inhibitors as in ( A ). Cells were lysed, the lysate was used to immunoprecipitate mTOR, and immunoprecipitates were analysed for proteins indicated. Alternatively, 30 μg of lysate was used for SDS/PAGE and immunoblot analysis of 4E-BP1 or tubulin (gel loading control). ( D ) HEK293T cells overexpressing FLAG–raptor were incubated with EBSS ± AAs as described in ( A ) above and with 100 nM rapamycin, 50 μM SB415286 or 1 μM Ku-0063794, as indicated. Cells were lysed and raptor was immunoprecipitated using anti-FLAG antibodies prior to immunoblotting with anti-mTOR and anti-FLAG antibodies.

Article Snippet: L6 rat skeletal muscle cells were grown as described previously in α-MEM containing 2% (v/v) FBS (Biosera) [ ].

Techniques: Incubation, Lysis, Stable Transfection, Expressing, shRNA, Western Blot, Inhibition, Phospho-proteomics, SDS Page, Control, Immunoprecipitation