Journal: Journal of Cell Communication and Signaling

Article Title: Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle

doi: 10.1007/s12079-020-00599-8

Figure Lengend Snippet: Lactate increased ROS generation, thereby augmenting autophagy in differentiated C2C12 myotubes. a Lactate treatment increased transcription of autophagy-related gene expression (two-way ANOVA: main effect of dose, Atg9, F = 6; Atg7, F = 22.9; Beclin 1, F = 12.3; Gabarapl1, F = 12.3; LC3B, F = 8.9; p62, F = 6.1; all p < 0.01; main effect of treatment, Atg9, F = 51; Atg7, F = 228; Beclin 1, F = 146; Gabarapl1, F = 135; LC3B, F = 89; p62, F = 44; treatment × dose interaction, Atg9, F = 5.7; Atg7, F = 22.7; Beclin 1, F = 12.2; Gabarapl1, F = 12.2; LC3B, F = 8.7; p62, F = 6.4; all p < 0.01) and b, c autophagic flux (two-way ANOVA: increase in LC3-II, main effect of dose, F = 7.2, p < 0.01; main effect of treatment, F = 52, p < 0.01; treatment × dose interaction, F = 5.1, p < 0.01; increase in LC3II/LC3I, main effect of dose, F = 38, p < 0.01; main effect of treatment, F = 209, p < 0.01: treatment × dose interaction, F = 35, p < 0.01; decrease in p62, main effect of dose, F = 4.8, p < 0.01; main effect of treatment, F = 74, p < 0.01; treatment × dose interaction, F = 10.1, p < 0.01) in a dose-dependent manner in C2C12 myotubes maintained in vehicle or lactate (2, 6, 10, and 20 mM, 8 h) compared to the control values (dashed lines). d In-gel profile for densitometric scanning analysis of LC3-I, LC3-II, and p62. e Representative images of H2DCFDA fluorescence. Lactate dose-dependently increased ROS generation and TBARS in differentiated C2C12 myotubes maintained in 2, 6, 10, and 20 mM lactate for 60 min e3-6 compared to the baseline values (vehicle), but exerted no effect in C2C12 myotubes co-incubated with 5 mM N-acetylcysteine (NAC) and lactate e7-10 (two-way ANOVA: ROS, main effect of dose, F = 12.3, p < 0.01; main effect of treatment, F = 42.1, p < 0.01; dose × treatment interaction, F = 8.4, p < 0.01; TBARS, main effect of dose, F = 4.5, p < 0.01, main effect of treatment, F = 4.5, p < 0.01; dose × treatment interaction, F = 2.2, p = 0.08). 500 mM H2O2 was used as a positive control. (f, g) Quantification of H2DCFDA fluorescence. Pre-treatment with NAC (5 mM, 2 h) deterred stimulatory effect of lactate on autophagy-related gene expression a (two-way ANOVA: main effect of dose, Atg9, F = 7.4; Atg7, F = 23.1; Beclin 1, F = 12.3; Gabarapl1, F = 11.6; LC3B, F = 7.7; p62, F = 6.9; all p < 0.01; main effect of treatment, Atg9, F = 13.9; Atg7, F = 187; Beclin 1, F = 71.7; Gabarapl1, F = 78.2; LC3B, F = 37.4; p62, F = 41; two-way ANOVA: treatment × dose interaction, Atg9, F = 2.0, p = 0.9; Atg7, F = 19.7; Beclin 1, F = 7.2; Gabarapl1, F = 7.5; LC3B, F = 5.6; p62, F = 3.1; all p < 0.01) and autophagic flux (b, c) (two-way ANOVA: main effect of dose, LC3B-I, F = 1.2; LC3B-II, F = 6.6; p62, F = 8.7; LC3B-II/LC3B-I, F = 38.7; all p < 001; Fig. 1b; main effect of treatment, LC3B-I, F = 46.9; LC3B-II, F = 31.2; p62, F = 50.4; LC3B-II/LC3B-I, F = 38.7; all P < 001; treatment × dose interaction, LC3B-I, F = 5; LC3B-II, F = 5.7; p62, F = 7.9; LC3B-II/LC3B-I, F = 34; all p < 001), suggesting lactate regulates autophagy in C2C12 myotubes with ROS participation. The values are mean ± SD. For autophagy-related gene expression, values expressed as mean ± SD of fold change from basal values (vehicle). For autophagy flux markers, the fold changes in protein levels were normalized for β-actin and relative to their expression in basal condition (vehicle). # Significant difference with basal values (P < 0.01), * Significant difference with lactate + NAC group (P < 0.01). N = 6 per group

Article Snippet: C2C12 mouse skeletal muscle cells were obtained from Pasteur Institute.

Techniques: Gene Expression, Control, Fluorescence, Incubation, Positive Control, Expressing

Journal: Journal of Cell Communication and Signaling

Article Title: Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle

doi: 10.1007/s12079-020-00599-8

Figure Lengend Snippet: Activation of ERK1/2 was required for lactate-induced regulation of autophagy through ROS. a1 In-gel profile for densitometric scanning analysis of p38-MAPK and ERK l/2 and their phosphorylated level in C2C12 myotubes maintained in vehicle or H2O2, or lactate 10 mM for 8 h in the absence or presence of NAC. a2 Lactate increased phosphorylataion of p38-MAPK (two-way ANOVA: main effect of treatment, F = 90.1 p < 0.01; dose × treatment interaction, F = 1.5, p = 0.23) and ERK l/2 (two-way ANOVA: main effect of treatment, F = 113 p < 0.01; dose × treatment interaction, F = 0.86, p = 0.43) compared to control values (dashed lines), but its effects were deterred in the presence of NAC (two-way ANOVA: main effect of treatment, p38-MAPK, F = 56.8, p < 0.01; dose × treatment interaction, F = 0.53, p = 0.59; main effect of treatment, P-Erk1/2, F = 64.2, p < 0.01; dose × treatment interaction, F = 0.44, p = 0.64). b1 In-gel profile and b2–4 bar plots for autophagy-related gene expression and autophagic flux in C2C12 cells maintained in vehicle or lactate (6, 10, and 20 mM, 8 h) in the absence or presence of ERK1/2 inhibitor U0126 (2.5 µM) and p38-MAPK inhibitor SB203580 (10 µM) for 8 h. ERKl/2 inhibition by U0126 abolished the stimulatory effect of lactate on autophagy-related gene expression (one-way ANOVA, all p < 0.01) Atg9, F = 4.7; Atg7, F = 32.8; Beclin 1, F = 31.5; Gabarapl1, F = 13.9; LC3B, F = 18.7; p62, F = 10; all p < 0.01) and autophagic flux (one-way ANOVA: LC3B-I, F = 52; LC3B-II, 12.9; p62, F = 21.7, LC3B-II/LC3B-I, F = 19.3; all p < 0.01), but p38-MAPK inhibition by SB203580 had no effect, suggesting lactate-induced autophagy regulation in C2C12 myotubes is mediated through activation of ERK1/2. * Significant difference between groups (P < 0.01). N = 6 per group

Article Snippet: C2C12 mouse skeletal muscle cells were obtained from Pasteur Institute.

Techniques: Activation Assay, Control, Gene Expression, Inhibition

Journal: Journal of Cell Communication and Signaling

Article Title: Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle

doi: 10.1007/s12079-020-00599-8

Figure Lengend Snippet: ERK1/2/mTOR/p70S6K pathway contributes to lactate-induced autophagy activation in differentiated C2C12 myotubes. a1–a3 Effects of U0126 (2.5 µM), Rapamycin (10 nM), or BI-D1870 (10 µM) on protein levels of ERK1/2/mTOR/p70S6K signaling pathway components in C2C12 cells maintained in the vehicle or lactate 10 mM for 8 h. Erk1/2 inhibition by U0126 deterred lactate effects on values of P-mTOR and p-p70S6K expression which restored them to their control values (dashed line). mTOR inhibition by SB203580 decreased p-p70S6K levels, p70S6K inhibition had no effect on P-ERK 1/2 and P-mTOR levels. a4 Autophagy-related gene expression and a5, 6 autophagic flux in C2C12 cells maintained in vehicle or lactate (6, 10, and 20 mM, 8 h) in the absence or presence of U0126, Rapamycin, or BI-D1870. For all protein measurements, fold changes in protein levels were normalized for β-actin and relative to their expression in basal condition (vehicle). * Significant difference with basal values (P < 0.01), + Significant difference with lactate + U0126 group (P < 0.01), ^ Significant difference with lactate + Rapamycin group (P < 0.01). N = 6 per group

Article Snippet: C2C12 mouse skeletal muscle cells were obtained from Pasteur Institute.

Techniques: Activation Assay, Inhibition, Expressing, Control, Gene Expression

Journal: Journal of Cell Communication and Signaling

Article Title: Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle

doi: 10.1007/s12079-020-00599-8

Figure Lengend Snippet: Changing the cellular redox associated to lactate oxidation is vital for ERK1/2-mediated autophagic effect of lactate on C2C12 myotubes. C2C12 cells maintained in the vehicle or lactate (2, 6, 10, and 20 mM, 8 h) in the absence or presence of 3-OBA (2 mM) or Oxamate (10 mM) for 8 h. a and b Only 20 mM lactate increased GPR81 gene and protein expression compared to the control values (dashed line). lactate oxidation inhibition by Oxamate abolished lactate stimulatory effect on autophagy-related gene expression c; one-way ANOVA, Atg9, F = 13.5; Atg7, F = 537; Beclin1, F = 34.3, Gabarapl1, F = 23.6; LC3B, F = 29.8, p62, F = 19.4; all p < 0.01), autophagic flux markers (d, e; one-way ANOVA: LC3B-II, F = 23.3; LC3B-II/LC3B-I, F = 142.1; LC3B-I, 32.5; p62, F = 19.4, all p < 0.01), and P-ERK 1/2 (f), inhibition of GPR81 by 3-OBA had no effect. The values expressed as mean ± SD of fold change from basal values (dashed line). For all protein measurement, fold changes in protein levels were normalized for β-actin and relative to their expression in basal condition (dashed line). * Significant difference with basal values (P < 0.01), # Significant difference with lactate + oxamate group (P < 0.01), N = 6 per group

Article Snippet: C2C12 mouse skeletal muscle cells were obtained from Pasteur Institute.

Techniques: Expressing, Control, Inhibition, Gene Expression

Journal: Journal of Cell Communication and Signaling

Article Title: Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle

doi: 10.1007/s12079-020-00599-8

Figure Lengend Snippet: TBARS (nmol MDA/µg protein) values in C2C12 myotubes incubated with different doses of lactate. H2O2 was used as a positive control

Article Snippet: C2C12 mouse skeletal muscle cells were obtained from Pasteur Institute.

Techniques: Incubation

Journal: Journal of Cell Communication and Signaling

Article Title: Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle

doi: 10.1007/s12079-020-00599-8

Figure Lengend Snippet: Changing cell’s redox balance (NAD +/NADH) resulting from lactate conversion to pyruvate augments reactive oxygen species (ROS) levels in differentiated C2C12 myotubes and mature skeletal muscle. The enhanced levels of ROS activate ERK1/2/mTOR/p70S6K signaling pathway, thereby increasing autophagy in skeletal muscle

Article Snippet: C2C12 mouse skeletal muscle cells were obtained from Pasteur Institute.

Techniques:

Journal: Cell Death & Disease

Article Title: Lipid droplets contribute myogenic differentiation in C2C12 by promoting the remodeling of the acstin-filament

doi: 10.1038/s41419-021-04273-8

Figure Lengend Snippet: A The schematic diagram of cell migration assay. C2C12 cells were seeded in the four wells in a 35-nm dish. After treatment, the culture insert was removed, then the dish was observed in a live cell workstation. B The images of cell migration at 0, 360, 1605, and 2655 min. The yellow arrows indicate the loaded cells under migration and the blue arrows indicate unloaded cells under migration. C Schematic diagram of counting the number of migrating cells. D The number of migrating cells at 24 and 48 h. E The microfilaments and LDs were marked in loaded and unloaded cells. The arrows indicate the pseudopodia in loaded cells and the arrow head indicate the pseudopodia in unloaded cells. F The number of filpopdia in E ( N = 30). * p < 0.05. Results are from three technical repeats ( N = 3) for a representative of three biological repeats ( N = 3).

Article Snippet: The C2C12 cell line was purchased from CoWin Biosciences (#CW2915F, Beijing, China).

Techniques: Cell Migration Assay, Migration

Journal: Cell Death & Disease

Article Title: Lipid droplets contribute myogenic differentiation in C2C12 by promoting the remodeling of the acstin-filament

doi: 10.1038/s41419-021-04273-8

Figure Lengend Snippet: A The rate of actin-filament remodeling. C2C12 cells were pre-treated with 400 μM of oleic acid (OA, named loaded cells) or 3% bovine serum albumin (BSA, control, named unloaded cells). For DGATi treatment, the inhibitor of DGAT1 and DGAT2 was added into medium for 12 h before OA treatment, bar, 20 μm. B The count of microfilament number per cell. * p < 0.05. Results are from three technical repeats ( N = 3) for a representative of three biological repeats ( N = 3).

Article Snippet: The C2C12 cell line was purchased from CoWin Biosciences (#CW2915F, Beijing, China).

Techniques: Control

Journal: Cell Death & Disease

Article Title: Lipid droplets contribute myogenic differentiation in C2C12 by promoting the remodeling of the acstin-filament

doi: 10.1038/s41419-021-04273-8

Figure Lengend Snippet: A Co-localization of ACTN3, microfilaments, and LDs in C2C12 cells. B The C2C12 cells were treated with cytochalasin D for 2 h. Then, the medium was replaced with a fresh medium. The LDs were isolated at 0 h, 0.5 h, 1 h, and 2 h, respectively. The ACTN3 levels were detected by Western blotting. C The LD-targeting ACTN3 plasmid was constructed by inserting a PAT domain (PLIN1 1–193aa) at the N-terminus of ACTN3. The co-localization of PAT-ACTN3-EGFP, LDs, and microfilaments in C2C12 cells was detected by a co-confocal microscope. The fluorescence intensity, along with the arrow line, is plotted in the below graphs. D The C2C12 cells (expressing PAT-ACTN3-EGFP) were treated with cytochalasin D for 2 h, then the medium was replaced with a fresh medium. The subcellular components were isolated at 0 and 1 h, respectively. The levels of EGFP were detected. ACTB was utilized as the microfilament reference protein and ATP1A was utilized as cytomembrane reference protein. * p < 0.05. Results are from three technical repeats ( N = 3) for a representative of three biological repeats ( N = 3).

Article Snippet: The C2C12 cell line was purchased from CoWin Biosciences (#CW2915F, Beijing, China).

Techniques: Isolation, Western Blot, Plasmid Preparation, Construct, Microscopy, Fluorescence, Expressing

Journal: Cell Death & Disease

Article Title: Lipid droplets contribute myogenic differentiation in C2C12 by promoting the remodeling of the acstin-filament

doi: 10.1038/s41419-021-04273-8

Figure Lengend Snippet: A The C2C12 cells were treated with cytochalasin D for 2 h, then the medium was replaced with a fresh medium. The co-localization of PAT-ACTN3-EGFP and ARF1-DsRed was detected. The fluorescence intensity, along with the arrow line, is plotted in the graph below. B The co-localization of LDs and ARF1-EGFP in C2C12 cells was detected. C The rate of actin-filament remodeling in loaded cells with brefeldin A treatment and unloaded cells, bar, 20 μm. D The count of microfilament number per cell. * p < 0.05. Results are from three technical repeats ( N = 3) for a representative of three biological repeats ( N = 3).

Article Snippet: The C2C12 cell line was purchased from CoWin Biosciences (#CW2915F, Beijing, China).

Techniques: Fluorescence

Journal: Burns & Trauma

Article Title: Enhancing diabetic muscle repair through W-GA nanodots: a nanomedicinal approach to ameliorate myopathy in type 2 diabetes

doi: 10.1093/burnst/tkae059

Figure Lengend Snippet: Evaluation of the biocompatibility of W-GA in vitro. ( a ) C2C12 cells cultured for 7 days in W-GA were evaluated for proliferation and cytotoxicity using live/dead staining. Scale bar = 500 μm. ( b ) Survival rate of C212 cells ( n = 3) is shown as the mean ± standard deviation. The statistical significance of differences between treatments was determined by one-way ANOVA and the Bonferroni posthoc correction. NS: Not significant. ( c ) C2C12 cell proliferation capacity was assessed 24 hours post-treatment using BrdU incorporation. The green signal represents BrdU. Scale bar = 500 μm. ( d ) Quantification of BrdU assay data ( n = 3). The data are presented as the mean ± standard deviation. NS: Not significant. ( e ) The cell proliferation ability of C2C12 cells in the W-GA group was further evaluated using a CCK-8 assay. The data are presented as the mean ± standard deviation. NS: Not significant

Article Snippet: C2C12 myoblasts (a mouse cell line) were obtained from ScienCell Research Laboratories and cultured in Dulbecco's modified Eagle’s medium (DMEM; containing 25 mM glucose; Gibco, USA), which included 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA), in a humidified atmosphere at 37°C and 5% CO 2 .

Techniques: In Vitro, Cell Culture, Staining, Standard Deviation, BrdU Incorporation Assay, BrdU Staining, CCK-8 Assay

Journal: Burns & Trauma

Article Title: Enhancing diabetic muscle repair through W-GA nanodots: a nanomedicinal approach to ameliorate myopathy in type 2 diabetes

doi: 10.1093/burnst/tkae059

Figure Lengend Snippet: Antiapoptotic, antioxidative, and myogenic differentiation-promoting effects of W-GA. ( a ) Flow cytometry profiles showing the abundance of total C2C12 cells under various treatment conditions, along with apoptosis events in C2C12 cells under different therapeutic interventions. ( b ) Quantification of flow cytometry data for apoptotic cells ( n = 3). The data are presented as the mean ± standard deviation. NS: Not significant, * * p < 0.01. The statistical significance of differences between treatments was determined by one-way ANOVA and the Bonferroni posthoc correction. ( c ) Flow cytometry profiles showing the production of ROS in C2C12 cells under different treatment conditions. ( d ) Quantification of flow cytometry data for ROS production ( n = 3). The data are presented as the mean ± standard deviation. NS: Not significant, * p < 0.05. ( e ) Representative immunofluorescence image illustrating MYHC and MyoD protein expression in C2C12 myoblasts. Scale bar = 100 μm. ( f ) Quantitative analysis and intergroup comparison of myotube diameters ( n = 3). The statistical significance of differences between treatments was determined by one-way ANOVA and the Bonferroni posthoc correction. NS: Not significant, * p < 0.05, * * p < 0.01

Article Snippet: C2C12 myoblasts (a mouse cell line) were obtained from ScienCell Research Laboratories and cultured in Dulbecco's modified Eagle’s medium (DMEM; containing 25 mM glucose; Gibco, USA), which included 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA), in a humidified atmosphere at 37°C and 5% CO 2 .

Techniques: Flow Cytometry, Standard Deviation, Immunofluorescence, Expressing, Comparison