Journal: Autophagy

Article Title: Mitophagy-mediated S1P facilitates muscle adaptive responses to endurance exercise through SPHK1-S1PR1/S1PR2 in slow-twitch myofibers

doi: 10.1080/15548627.2025.2488563

Figure Lengend Snippet: Mitophagic degradation of ceramides during exercise generates S1P and facilitates mitochondrial biogenesis through SPHKs-S1P-S1PRs in skeletal muscles. (A) Schematic representation of the ceramide-S1P metabolism. (B) Quantification of sphingosine, S1P and ceramide concentrations in whole fraction and mitochondrial fraction of slow-twitch muscles (RG) from WT mice at rest (sed), during exercise (ex) and recovering for 3 h after exercise (rec) (n = 4–6). (C) Representative immunofluorescence images and Pearson’s correlation coefficients of ceramide (green), MitoTracker (red) and LC3 (magenta) in C2C12 cells treated with or without 10 μM FCCP (n = 12–16 cells from 3 biological replicates for every group). Scale bar: 2 μm. (D) Quantification of sphingosine, S1P and ceramide concentrations in whole fraction and mitochondrial fraction of slow-twitch muscles (RG) from fundc1 KO mice at rest (sed), during exercise (ex) and recovering for 3 h after exercise (rec) (n = 4–5). (E) quantification of sphingosine, S1P and ceramide concentrations in whole fraction and mitochondrial fraction of C2C12 myotubes treated with 10 μM FCCP, in the absence or presence of 50 μM CQ pre-treatment (n = 4–5). (F) Quantification of S1P concentrations in C2C12 myotubes treated with 10 μM FCCP for 4 h with/without 50 μM NOE or 5 μM SKI-II pre-treatment (n = 4). (G) PPARGC1A protein levels in the C2C12 myotubes 48 h after the stimulation of 10 μM FCCP with/without 50 μM CQ in the absence or presence of 5 μM S1P (n = 5). (H) PPARGC1A protein levels in the C2C12 myotubes 48 h after the stimulation of 10 μM FCCP with/without 50 μM NOE in the absence or presence of 5 μM S1P (n = 3). (I) PPARGC1A protein levels in the C2C12 myotubes 48 h after the stimulation of 10 μM FCCP with/without 5 μM SKI-II in the absence or presence of 5 μM S1P (n = 4). (J) PPARGC1A protein levels in si NC , si S1pr1 , si S1pr2 or si S1pr3 transfected C2C12 myotubes 48 h after the stimulation of 10 μM FCCP (n = 4). (K) PPARGC1A protein levels in C2C12 myotubes treated with 10 μM JTE-013 for 48 h after the stimulation of 10 μM FCCP (n = 4). (L) Mitochondrial turnover ratio of si NC , si S1pr1 , si S1pr2 or si S1pr3 transfected C2C12 myotubes 48 h after the stimulation of 10 μM FCCP (n = 3). (M) Mitochondrial turnover ratio of C2C12 myotubes treated with 10 μM JTE-013, 5 μM W146 or 10 μM CAY10444 for 48 h after the stimulation of 10 μM FCCP (n = 3). All data are shown as the means ± SEM. Differences between two groups were assessed using two-tailed student’s t tests (C). Differences for more than two groups were assessed using one-way ANOVA with Tukey’s post hoc test (B, D-M). p values are indicated as follows: * p < 0.05, ** p < 0.01. *** p < 0.001, **** p < 0.0001.

Article Snippet: Differentiated C2C12 myotubes were treated with 10 μM FCCP (MCE, HY-100410) for 4 h after pretreatment with 50 μM CQ (Sigma-Aldrich, C6628) for 4 h, 50 μM NOE (MCE, HY-107542) for 24 h or 5 μM SKI-II (MCE, HY-13822) for 24 h. Then, the FCCP was removed, and the cells were incubated with 10 μM S1P, 5 μM W146, 10 μM JTE-013 or 10 μM CAY10444 for 48 h.

Techniques: Muscles, Immunofluorescence, Transfection, Two Tailed Test

Journal: PLOS One

Article Title: Protopanaxadiol stimulates glucose consumption by modulating the AMP-activated protein kinase pathway in myotubes, hepatoma cells, and adipocytes

doi: 10.1371/journal.pone.0328486

Figure Lengend Snippet: Effects of (A) protopanaxadiol (PPD), (B) Rg3, (C) Rc, and (D) Rh2 on the viability of C2C12 cells compared to that of the untreated control, as determined via the Ez-Cytox cell viability assay for 24 h. Inhibitory effects of ginsenosides and rosiglitazone on lipid accumulation in C2C12 cells. Glucose consumption in C2C12 cells treated with palmitic acid (0.25 mM) for 24 h with or without pre-treatment with the indicated concentration of (E) PPD, (F) Rg3, (G) Rc, (H) Rh2, and insulin assessed via glucose uptake assay using 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-Deoxyglucose (2-NBDG) (n = 3 independent experiments, * p < 0.05, Kruskal–Wallis non-parametric test). Data are represented as the mean ± SEM.

Article Snippet: C2C12 myotubes (ATCC CRL-1772), HepG2 hepatoma cells (ATCC HB-8065), and 3T3-L1 adipocytes (ATCC CL-173) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen Co., Grand Island, NY, USA) in a humidified atmosphere (37 °C, 95% air, 5% CO 2 ).

Techniques: Control, Viability Assay, Concentration Assay

Journal: PLOS One

Article Title: Protopanaxadiol stimulates glucose consumption by modulating the AMP-activated protein kinase pathway in myotubes, hepatoma cells, and adipocytes

doi: 10.1371/journal.pone.0328486

Figure Lengend Snippet: (A–H) Protein expression levels and ratios of band intensities of insulin receptor substrate-1 (IRS-1), p-IRS-1, Akt, p-Akt, AMP-activated protein kinase α (AMPKα), p-AMPKα, glycogen synthase kinase-3β (GSK-3β), p-GSK-3β, glycogen synthase, p-glycogen synthase, glucose-6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase1/Phosphoenolpyruvate carboxylase (PCK1/PEPC) in C2C12 cells treated with palmitic acid (0.25 mM) for 24 h with or without pre-treatment with the indicated concentration of PPD (n = 3 independent experiments, * p < 0.05, Kruskal–Wallis non-parametric test). Data are represented as the mean ± SEM.

Article Snippet: C2C12 myotubes (ATCC CRL-1772), HepG2 hepatoma cells (ATCC HB-8065), and 3T3-L1 adipocytes (ATCC CL-173) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen Co., Grand Island, NY, USA) in a humidified atmosphere (37 °C, 95% air, 5% CO 2 ).

Techniques: Expressing, Concentration Assay