Journal: The international journal of biochemistry & cell biology

Article Title: Maduramicin inactivation of Akt impairs autophagic flux leading to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells

doi: 10.1016/j.biocel.2019.105573

Figure Lengend Snippet: Mad induces apoptosis in skeletal myoblast cells in a concentration-dependent manner. C2C12 and L6 cells were treated with Mad (0–1 μM) for 24 h. (A) Apoptotic cells were evaluated by nuclear fragmentation and condensation (arrows) using DAPI staining (upper panel) and concurrently by in situ detection of fragmented DNA (in green) using TUNEL staining (lower panel). Scale bar: 20 μm. (B) The percentage of cells with fragmented nuclei and the number of TUNEL-positive cells were quantified. (C) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (D) The blots for cleaved-caspase-3 and cleaved-PARP were semi-quantified. (E) Caspase-3/7 activities were detected using Caspase-3/7 Assay Kit. Results are presented as mean ± SEM (n = 3–5). *p < 0.05, P < 0.01, difference with control group.

Article Snippet: Cell culture Mouse C2C12 myoblast cells (C2C12 cells) (#CRL-1772) and rat L6 myoblast cells (L6 cells) (#CRL-1458) were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Concentration Assay, Staining, In Situ, TUNEL Assay, Western Blot, Control

Journal: The international journal of biochemistry & cell biology

Article Title: Maduramicin inactivation of Akt impairs autophagic flux leading to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells

doi: 10.1016/j.biocel.2019.105573

Figure Lengend Snippet: Mad triggers an increase in autophagosomes with a concomitant elevation of LC3-II and p62 in skeletal myoblast cells. C2C12 and L6 cells, or C2C12 and L6 cells infected with Ad-GFP-LC3, respectively, were treated with Mad (0–1 μM) for 24 h. (A) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) The blots for LC3-II and p62 were semi-quantified. (C and D) The cells were labeled using a specific autophagolysosome marker MDC staining and then the fluorescence intensity (in green) for MDC-labeled vacuoles was imaged (C) and quantified (D) as described in Materials and Methods. Scale bar: 20 μm. (E and F) Shown are representative GFP-LC3 fluorescence images (in green) (E) and quantified number for GFP-LC3 puncta (F) in the cells. Scale bar: 2 μm. Results are presented as mean ± SEM (n = 3–5). *p < 0.05, **p < 0.01, difference with control group.

Article Snippet: Cell culture Mouse C2C12 myoblast cells (C2C12 cells) (#CRL-1772) and rat L6 myoblast cells (L6 cells) (#CRL-1458) were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Infection, Western Blot, Control, Labeling, Marker, Staining, Fluorescence

Journal: The international journal of biochemistry & cell biology

Article Title: Maduramicin inactivation of Akt impairs autophagic flux leading to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells

doi: 10.1016/j.biocel.2019.105573

Figure Lengend Snippet: Mad inactivation of Akt links impaired autophagic flux to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells. C2C12 and L6 cells, or C2C12 and L6 cells infected with Ad-GFP-LC3, respectively, were treated with Mad (0–1 μM) for 24 h, or pretreated with/without Akt inhibitor X (10 μM) for 2 h and then treated with/without Mad (0.5 and 1 μM) for 24 h. (A and C) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B and D) The blots for p-Akt (Ser473), p-Akt (Thr308), p-GSK3β (Ser9), LC3-II, p62 and cleaved-caspase-3 were semi-quantified. (E) The number of GFP-LC3 punctate structures per cell was counted and calculated. (F) The cell viability was determined by the MTS assay. (G) Apoptotic cells were evaluated by nuclear fragmentation and condensation using DAPI staining. Results are presented as mean ± SEM (n = 3–5). ap < 0.05, difference with control group; bp < 0.05, difference with 0.5 μM Mad group; cp < 0.05, difference with 1 μM Mad group.

Article Snippet: Cell culture Mouse C2C12 myoblast cells (C2C12 cells) (#CRL-1772) and rat L6 myoblast cells (L6 cells) (#CRL-1458) were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Infection, Western Blot, Control, MTS Assay, Staining

Journal: The international journal of biochemistry & cell biology

Article Title: Maduramicin inactivation of Akt impairs autophagic flux leading to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells

doi: 10.1016/j.biocel.2019.105573

Figure Lengend Snippet: Mad elicits impaired autophagic flux leading to autophagosome-dependent apoptosis in skeletal myoblast cells. C2C12 and L6 cells, or C2C12 and L6 cells infected with Ad-mCherry-GFP-LC3 or Ad-GFP-LC3, respectively, were treated with/ without Mad (0.5 and 1 μM) for 24 h, or pretreated with/without CQ (5 μM) for 1 h and then treated with/without Mad (0.5 and 1 μM) for 24 h. (A) Co-localization of both GFP and mCherry fluorescence for GFP+/mCherry+-LC3 (yellow) puncta was shown in the cells. Scale bar: 2 μm. (B) GFP+/mCherry+-LC3 puncta per cell were quantified. (C) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (D) The blots for LC3-II, p62, and cleaved-caspase-3 were semi-quantified. (E) The fluorescence intensity for MDC-labeled vacuoles in the cells was quantified. (F) The number of GFP-LC3 punctate structures per cell was counted and calculated. (G) Apoptotic cells were evaluated by nuclear fragmentation and condensation using DAPI staining. Results are presented as mean ± SEM (n = 3–5). ap < 0.05, difference with control group; bp < 0.05, − CQ group versus + CQ group.

Article Snippet: Cell culture Mouse C2C12 myoblast cells (C2C12 cells) (#CRL-1772) and rat L6 myoblast cells (L6 cells) (#CRL-1458) were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Infection, Fluorescence, Western Blot, Control, Labeling, Staining

Journal: The international journal of biochemistry & cell biology

Article Title: Maduramicin inactivation of Akt impairs autophagic flux leading to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells

doi: 10.1016/j.biocel.2019.105573

Figure Lengend Snippet: Rapamycin rescues Mad-impaired autophagic flux from accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells. C2C12 and L6 cells, or C2C12 and L6 cells infected with Ad-GFP-p62 or Ad-GFP-LC3, respectively, were pretreated with/without rapamycin (Rapa, 0.2 μg/ml) for 24 h and then treated with/without Mad (0.5 and 1 μM) for 24 h. (A) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) The blots for LC3-II, p62, and cleaved-caspase-3 were semi-quantified. (C and D) Shown were representative GFP-p62 fluorescence images (in green) (C) and quantified number for GFP-p62 (D) in the cells. Scale bar: 2 μm. (E) The number of GFP-LC3 punctate structures per cell was counted and calculated. (F) Apoptotic cells were evaluated by nuclear fragmentation and condensation using DAPI staining. Results are presented as mean ± SEM (n = 3–5). ap < 0.05, difference with control group; bp < 0.05, difference with 0.5 μM Mad group; cp < 0.05, difference with 1 μM Mad group.

Article Snippet: Cell culture Mouse C2C12 myoblast cells (C2C12 cells) (#CRL-1772) and rat L6 myoblast cells (L6 cells) (#CRL-1458) were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Infection, Western Blot, Control, Fluorescence, Staining

Journal: Diabetologia

Article Title: Treatment with a β-2-adrenoceptor agonist stimulates glucose uptake in skeletal muscle and improves glucose homeostasis, insulin resistance and hepatic steatosis in mice with diet-induced obesity

doi: 10.1007/s00125-020-05171-y

Figure Lengend Snippet: In vitro and acute in vivo effects of clenbuterol. ( a ) Clenbuterol stimulation of L6 myotubes induced glucose uptake in a dose-dependent manner; n = 5–8; data were analysed by one-way ANOVA with Dunnett’s multiple comparison test. ( b , c ) Stimulation of L6 myotubes stably expressing GLUT4myc with 1 μmol/l clenbuterol induces GLUT4 translocation as quantified in ( c ); n = 3; data were analysed with unpaired two-tailed Student’s t test. Scale bars, 50 μm. ( d ) In vivo glucose uptake in gastrocnemius muscle of chow-fed mice treated with 1 mg/kg clenbuterol for 1 h; n = 5–6; data were analysed with unpaired two-tailed Student’s t test. ( e , f ) Acute effects of clenbuterol on blood glucose ( e ) and plasma insulin ( f ); n = 6 (diet-induced obesity was developed in C57Bl/6N mice maintained at 30°C and on HFD for 7 months; mice were fasted in the morning for 6 h prior to clenbuterol, glucose or saline i.p. injection); data were analysed by two-way ANOVA with Dunnett’s multiple comparison test. In all graphs: * p < 0.05, ** p < 0.01, *** p < 0.001 vs vehicle (in e and f vs vehicle at the same time point)

Article Snippet: L6 rat myoblasts and L6 myoblasts stably expressing GLUT4myc were purchased from KeraFast (ESK201 and ESK202), where they were tested for mycoplasma.

Techniques: In Vitro, In Vivo, Stable Transfection, Expressing, Translocation Assay, Two Tailed Test, Injection