Supplementary Figure 1

Effects of RSV, R3G and R4G treatment on cellular glucose uptake in insulin-resistant primary mouse hepatocytes and differentiated C2C12 cells. (A) Hepatocytes were isolated from C57BL/6JSlc mice1. After cells attachment, hepatocytes were serum-starved for 12 h followed by treatment with 1×10-6 mol/L of insulin for 24 h. Then, the cells were cultured with 1 µM of RSV, R3G and R4G in the presence of 1×10-6 mol/L of insulin for another 24 h. The decreased cellular glucose uptake was notably prevented by 1 µM of RSV, R3G and R4G treatments by 26.6%, 4.17% and 15.9% in the primary mouse hepatocytes, respectively. (B) C2C12 myocytes, obtained from American Type Culture Collection (ATCC, Manassas, VA), were maintained in DMEM containing 10% FBS in an atmosphere of 5% CO2 at 37 °C. For differentiation into myotubes, confluent cells were cultured in DMEM supplemented with 2% horse serum for 5 days. The cells were treated with 1×10-6 mol/L of insulin for 24 h. Then, the cells were cultured with 1 µM of RSV, R3G and R4G in the presence of 1×10-6 mol/L of insulin for another 24 h. The decreased cellular glucose uptake was prevented by 1 µM of RSV, R3G and R4G treatments by 7.8%, 1.7% and 3.9% in the differentiated C2C12 cells, respectively. Values shown are means ± SD from three separate determinations. * p < 0.05; # p < 0.05 vs. only insulin-resistant group.
Supplementary Figure 2

Effects of RSV, R3G and R4G treatment on the expressions of p-Akt Ser473 and Akt in insulin-resistant HepG2 cells.
(A) Cells were incubated with 1 × 10-6 mol/L insulin for 24 h. The left side of panel of A showed representative western blot for phosphorylated and total levels of Akt, while the right side depicted quantitative analysis of each band. (B) After cells were incubated with 1 × 10-6 mol/L insulin for 24h, their media was discarded and were grown in the presence of medium containing 1µM of RSV, R3G and R4G for another 24 h, respectively. Similarly, left side of panel of A showed representative western blot for phosphorylated and total levels of Akt, while the right side depicted quantitative analysis of each band. Values shown are means ± SD from three separate determinations. * p < 0.05; # p < 0.05 vs. only insulin-resistant HepG2 cells. 

Refenrence:

1.	S. Inokuchi, T. Aoyama, K. Miura, C. H. Osterreicher, Y. Kodama, K. Miyai, S. Akira, D. A. Brenner and E. Seki, Proceedings of the National Academy of Sciences of the United States of America, 2010, 107, 844-849.