Fig. 1Effects of Rg3 on the insulin signaling pathway. In C2C12 myotubes, 100 µM Rg3 was administered for 24 hours with or without (A) 500 µM palmitate or (B) 10 µM antimycin A. Experiment was repeated four times. pIRS-1, phosphorylated insulin receptor substrate-1; IRS-1, insulin receptor substrate-1.
Fig. 2Effects of Rg3 on mitochondrial function. In C2C12 myotubes, 100 µM Rg3 was administered for 24 hours with or without 10 µM antimycin A. (A) Cellular adenosine triphosphate (ATP) levels. (B) Representative graphs of oxygen consumption rate (OCR). OCR was measured under the basal conditions, following the addition of oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and antimycin A/rotenone. (C) Basal OCR. Each experiment was repeated more than four times. Data was presented as mean±standard error of mean. aP<0.05.
Fig. 3Effects of Rg3 on the expression of key genes involved in mitochondrial biogenesis. In C2C12 myotubes, 100 µM Rg3 was administered for 24 hours with or without 10 µM antimycin A. (A) Expression of mitochondrial complexes by Western blot. (B) mRNA expression of mitochondrial complexes by quantitative reverse transcription polymerase chain reaction (RT-PCR). (C) mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor (Tfam) by quantitative RT-PCR. RT-PCR was performed in duplicate, and each experiment was repeated five times. aP<0.05.
Fig. 4Effects of Rg3 and adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway. After pretreatment of 10 µM antimycin A, 100 µM Rg3 was administered for 24 hours with or without 10 µM compound C in C2C12 myotubes. The expression of mitochondrial complexes and AMPK was evaluated by Western blot. pAMPK, phosphorylated AMPK.
Table 1Primer sequences used for quantitative reverse transcription polymerase chain reaction
|
Forward |
Reverse |
Complex I (20 kDa) |
5'-CCAGCTGCGCAGAGTTCATC-3' |
5'-GAGAGAGCTTGGGGACCACG-3' |
Complex II (Ip 30 kDa) |
5'-TCTACCGCTGCCACACCATC-3' |
5'-AAGCCAATGCTCGCTTCTCC-3' |
Complex III (Core2) |
5'-CCATTGGAAATGCAGAGGCA-3' |
5'-GGCTGGTGACTTCCTTTGGC-3' |
Complex IV (COXII) |
5'-TCATGAGCAGTCCCCTCCCT-3' |
5'-GCCATAGAATAACCCTGGTCGG-3' |
Complex V (F1α) |
5'-ATCTATGCGGGTGTACGGGG-3' |
5'-AGGGACTGGTGCTGGCTGAT-3' |
PGC-1α |
5'-ACCTGACACAACGCGGACAG-3' |
5'-TCTCAAGAGCAGCGAAAGCG-3' |
NRF-1 |
5'-AGGGCGGTGAAATGACCATC-3' |
5'-CGGCAGCTTCACTGTTGAGG-3' |
Tfam |
5'-GGGTATGGAGAAGGAGGCCC-3' |
5'-TCCCTGAGCCGAATCATCCT-3' |