Journal: Journal of Diabetes and Metabolic Disorders

Article Title: Effect of sub-toxic chlorpyrifos on redox sensitive kinases and insulin signaling in rat L6 myotubes

doi: 10.1007/s40200-018-0379-x

Figure Lengend Snippet: Bar diagram showing the effect of Chlorpyrifos exposure for 18hrs on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in L6 myoblast derived myotubes after normalized with beta actin by western blot hybridization. Data shown are means with standard errors of three independent experiments. C+i = Control with insulin, CPF6+i = Chlorpyrifos (6mg/L) exposure for 18 hrs followed by treatment with insulin (1uM for 20min), CPF12+i = Chlorpyrifos 12mg/L exposure for 18hrs followed by treatment with insulin (1uM for 20min).†p<0.05 in comparison to control, *p<0.05 in comparison to Chlorpyrifos 6mg/L exposure groups by one way ANOVA followed by Tukey HSD post-hoc test

Article Snippet: Rat skeletal myoblast (L6) cell line was obtained from the National Centre for Cell Sciences (Pune, India) and propagated at 37 °C in 5% CO 2 incubator in DMEM supplemented with 100 units/ml of penicillin G, 100 μg/ml of streptomycin sulfate, 0.25 μg/ml amphotericin B and 10% FBS.

Techniques: Phospho-proteomics, Derivative Assay, Western Blot, Hybridization, Control, Comparison

Journal: Journal of Diabetes and Metabolic Disorders

Article Title: Effect of sub-toxic chlorpyrifos on redox sensitive kinases and insulin signaling in rat L6 myotubes

doi: 10.1007/s40200-018-0379-x

Figure Lengend Snippet: Bar diagram showing the effect of Chlorpyrifos exposure for 18hrs on the serine phosphorylation of Akt in L6 myoblast derived myotubes after normalized with beta actin by western blot hybridization. Data shown are means with standard errors of three independent experiments. C+i = Control with insulin, CPF6+i = Chlorpyrifos (6mg/L) exposure for 18 hrs followed by treatment with insulin (1uM for 20min), CPF12+i = Chlorpyrifos 12mg/L exposure for 18hrs followed by treatment with insulin (1uM for 20min). †p<0.05 in comparison to control, *p<0.05 in comparison to Chlorpyrifos 6mg/L exposure groups by one way ANOVA followed by Tukey HSD post-hoc test

Article Snippet: Rat skeletal myoblast (L6) cell line was obtained from the National Centre for Cell Sciences (Pune, India) and propagated at 37 °C in 5% CO 2 incubator in DMEM supplemented with 100 units/ml of penicillin G, 100 μg/ml of streptomycin sulfate, 0.25 μg/ml amphotericin B and 10% FBS.

Techniques: Phospho-proteomics, Derivative Assay, Western Blot, Hybridization, Control, Comparison

Journal: Journal of Diabetes and Metabolic Disorders

Article Title: Effect of sub-toxic chlorpyrifos on redox sensitive kinases and insulin signaling in rat L6 myotubes

doi: 10.1007/s40200-018-0379-x

Figure Lengend Snippet: Bar diagram showing the effect of Chlorpyrifos exposure for 18hrs on the phosphorylation of p38 MAPK in L6 myoblast derived myotubes after normalized with beta actin by western blot hybridization. Data shown are means with standard errors of three independent experiments. C+i = Control with insulin, CPF6+i = Chlorpyrifos (6mg/L) exposure for 18 hrs followed by treatment with insulin (1uM for 20min), CPF12+i = Chlorpyrifos 12mg/L exposure for 18hrs followed by treatment with insulin (1uM for 20min). †p<0.05 in comparison to control, *p<0.05 in comparison to Chlorpyrifos 6mg/L exposure groups by one way ANOVA followed by Tukey HSD post-hoc test

Article Snippet: Rat skeletal myoblast (L6) cell line was obtained from the National Centre for Cell Sciences (Pune, India) and propagated at 37 °C in 5% CO 2 incubator in DMEM supplemented with 100 units/ml of penicillin G, 100 μg/ml of streptomycin sulfate, 0.25 μg/ml amphotericin B and 10% FBS.

Techniques: Phospho-proteomics, Derivative Assay, Western Blot, Hybridization, Control, Comparison

Journal: Journal of Diabetes and Metabolic Disorders

Article Title: Effect of sub-toxic chlorpyrifos on redox sensitive kinases and insulin signaling in rat L6 myotubes

doi: 10.1007/s40200-018-0379-x

Figure Lengend Snippet: Bar diagram showing the effect of Chlorpyrifos exposure for 18hrs on the phosphorylation of IκBα in L6 myoblast derived myotubes after normalized with beta actin by western blot hybridization. Data shown are means with standard errors of three independent experiments. C+i = Control with insulin, CPF6+i = Chlorpyrifos (6mg/L) exposure for 18 hrs followed by treatment with insulin (1uM for 20min), CPF12+i = Chlorpyrifos 12mg/L exposure for 18hrs followed by treatment with insulin (1uM for 20min). †p<0.05 in comparison to control, *p<0.05 in comparison to Chlorpyrifos 6mg/L exposure groups by one way ANOVA followed by Tukey HSD post-hoc test

Article Snippet: Rat skeletal myoblast (L6) cell line was obtained from the National Centre for Cell Sciences (Pune, India) and propagated at 37 °C in 5% CO 2 incubator in DMEM supplemented with 100 units/ml of penicillin G, 100 μg/ml of streptomycin sulfate, 0.25 μg/ml amphotericin B and 10% FBS.

Techniques: Phospho-proteomics, Derivative Assay, Western Blot, Hybridization, Control, Comparison

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

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Studies on the effects of hypothermia combined with hypoxia on rat skeletal muscle and lipid metabolism based on AMPK/PGC1α pathway

doi: 10.1186/s13018-021-02861-0

Figure Lengend Snippet: Effects of AMPK on the proliferation activity of rat skeletal muscle myoblasts. a Inhibition rate of cell proliferation. b The protein of skeletal muscle cells L6. A: Blank group. B: Hypoxic group. C: Hypoxic with AMPK inhibitor group. D: Hypothermic group. E: Hypothermic with AMPK inhibitor group. F: Hypoxic hypothermic group. G: Hypoxic hypothermic with AMPK inhibitor group. The results were presented as the mean ± standard deviation. * P < 0.05 and ** P < 0.01, *** P < 0.001 compared with the blank group (A)

Article Snippet: Skeletal muscle myoblast cells of rat L6 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Activity Assay, Inhibition, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)–mediated glucose uptake into skeletal muscle cells

doi: 10.1074/jbc.M117.801340

Figure Lengend Snippet: N-WASP inhibition blunts insulin-stimulated glucose uptake and GLUT4 translocation. A, L6-GLUT4myc myoblasts were treated with vehicle (DMSO) or 10 μm WISK and stimulated with 100 nm insulin for 20 min simultaneously. Cells were fixed and left unpermeabilized for labeling with anti-myc antibody. The immunofluorescent intensity of cell surface GLUT4 was normalized to the nucleic acid staining dye Syto 60. Values are means ± S.D. from three independent sets of cells. **, p < 0.01. B, L6-GLUT4myc myoblasts were treated with 0, 5, 10, 20, or 25 μm WISK together with 100 nm insulin for a total of 20 min. Cells were fixed and left unpermeabilized for labeling with anti-myc antibody. The immunofluorescent intensity of cell surface GLUT4 was normalized to the nucleic acid staining dye Syto 60. Values are means ± S.D. from three independent sets of cells. ***, p < 0.001. C, L6-GLUT4myc myotubes were used for 2-deoxyglucose uptake assays. Values are means ± S.D. from three independent sets of cells. *, p < 0.05. D and E, L6-GLUT4myc myoblasts were treated with DMSO or WISK, and the cellular ATP levels and percentage of viable cells were determined, respectively. Values are means ± S.D. from three independent sets of cells. **, p < 0.01; ***, p < 0.001.

Article Snippet: Materials Rat L6 GLUT4-myc skeletal muscle cells expressing c-myc–tagged GLUT4 protein were purchased from Kerafast and cultured as described previously ( 48 ).

Techniques: Inhibition, Translocation Assay, Labeling, Staining