Fig 5. 2-ME stimulates Akt/eNOS activation via PPARγ in endothelial cells.

A. HUVECs were exposed to 2-ME (10⁻⁵ M) for 20 min, in the presence or absence of GW9662 (GW—20 μM). Cell content of phosphor-Akt (P-Akt), phospho-eNOS (P-eNOS) or β-actin were shown by western blot. n = 3, * P < 0.01 vs. CON; # P < 0.01 vs.2-ME. B. HUVECs were exposed to 2-ME (10⁻⁵ M) for 20 min, in the presence or absence of GW9662 (GW—20 μM). The medium NO concentrations were measured. n = 5, * P < 0.01 vs. CON; # P < 0.01 vs.2-ME. C. Expression of PPARγ mRNA was detected by RT-PCR using total RNA extracted from HUVECs. GAPDH mRNA was used as an internal control. D. HUVECs were transfected with 100 nM target siRNAs for PPARγ or scrambled siRNA for 48 h and then treated with 2-ME (10⁻⁵ M) for 20 min. Cell content of P-eNOS, eNOS, P-Akt, Akt, PPARγ or β-actin were shown by western blot. n = 3, * P < 0.01 vs. corresponding CON; # P < 0.01 vs. corresponding 2-ME treatment group transfected with scrambled siRNA. E. HUVECs were treated with 2-ME at different doses as indicated for 20 min and then cells were stained with an Ab vs. tubulin (FITC; green staining). Nuclei were counterstained in blue with DAPI. All the experiments were repeated three times with consistent results, and a representative result is shown.F. The effect of GW9662 (GW—20 μM) on 2-ME (10⁻⁵ M)-induced dilation were shown. The experiments were repeated six times with consistent results.