Fig. 1(A) Total RNA was prepared for these cells after high glucose culture, and reverse transcription-polymerase chain reaction (RT-PCR) was conducted using specific Vav3 primers. PCR products were then run on 1% agarose gels and visualized under ultraviolet light. β-Actin mRNA was employed as a positive control. (B) C2C12 cells were stimulated at the indicated times with high glucose concentrations. The cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 antibody. Blotting with anti-β-actin antibody was used as a protein loading control. The results shown are from three independent experiments.
Fig. 2(A) Total RNA was cultured under high glucose culture conditions in the presence or absence of 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR) treatment, and reverse transcription-polymerase chain reaction (RT-PCR) was conducted using specific Vav3 primers. PCR products were then run on 1% agarose gels and visualized under ultraviolet light. β-Actin mRNA was employed as a positive control. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). The cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 antibody. Blotting with anti-β-actin antibody was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPK antibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. (D) C2C12 cells were cultured under high glucose conditions in the presence or absence of metformin. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPKantibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. The results shown are from three independent experiments.
Fig. 3(A) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMPK agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). Total cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-PAK and anti-PAK antibodies. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-paxillin and anti-paxillin antibodies was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence of metformin. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-PAK and anti-phospho-paxillin antibodies was performed. Blotting with anti-PAK and anti-paxillin antibodies was conducted as a protein loading control. The results shown are from three independent experiments.
Fig. 4(A) C2C12 cells were transiently transfected with 50 nM siRNA Vav3 for 2 days. Total cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 and anti-β-actin antibodies. (B) C2C12 cells were allowed to differentiate for 7 days. Cells were transiently transfected with Vav3 siRNA for 48 hours and then treated with metformin for 16 hours either in the presence or absence of compound C. Glucose uptake was measured using 2-deoxy-[3H]-d-glucose. The results shown are from three independent experiments. (C) Myoblasts stably expressing L6-GLUT4myc were differentiated and were transiently transfected with Vav3 siRNA and maintained for 48 hours, then incubated with metformin for 1 hour. Cell surface expression of GLUT4myc was detected using an antibody-coupled colorimetric absorbance assay. Results are displayed as the mean±SEM from three experiments. aP<0.05 for the basal values; bP<0.05 for the metformin-treated condition; cP<0.05 for the metformin plus Vav3 siRNA; dP<0.05 compared with metformin-treated condition.