Figure 3.

Effect of β-actin silencing on eNOS expression in cultured HAECs. HAECs were cultured up to 70% to 80% confluence. The β-actin specific siRNA was transfected to HAECs using Lipofectamine. For dose-dependent experiment, endothelial RNA and protein samples were collected after 24 or 48 hours of the siRNA treatment, respectively. We used a siRNA with the sequence homologous to a section of the luciferase coding region (sense 5′-GUCUGACAGUUACCAAUGC-3′, antisense 5′-GCAUUGGUAACUGUCAGAC-3′) as the nonspecific control because no luciferase was expressed in endothelial cells. A, Dose-dependent effect of β-actin siRNA on eNOS mRNA expression by the quantitative real-time reverse-transcription polymerase chain reaction. B, Dose-dependent effect of β-actin siRNA on eNOS protein levels by Western blotting. C, Time-dependent changes in eNOS mRNA levels when endothelial cells were treated with 50 nM β-actin siRNA. D, Time-dependent changes in eNOS protein levels when endothelial cells were treated with 50 nM β-actin siRNA. The levels of GAPDH mRNA or protein were used as the endogenous control for the adjustment of the eNOS mRNA and protein levels in cells treated with β-actin siRNA. **P < 0.01 by independent Student t test when compared with the endothelial cells treated with the luciferase specific siRNA as a negative control.