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. 2011 Dec;179(6):2740–2750. doi: 10.1016/j.ajpath.2011.08.011

Figure 2.

Figure 2

Increased GGPPS transcription under 10% cigarette smoke extract (CSE) treatment was dependent on EGR-1. A: Western blot analysis of EGR-1 and GGPPS during the time course of 10% CSE treatment. Beas-2B cells were treated with continuous 10% CSE until the samples were collected at the indicated times (upper panel). The EGR-1 level increased within 30 minutes after CSE treatment and decayed after 4 hours, and GGPPS increased after EGR-1 at 1 hour. In addition, Beas-2B cells were pretreated with CSE for only 5 minutes and then returned to the normal growth medium. The cells were lysed at the indicated times and subjected to Western blotting (lower panel). The EGR-1 level increased from 10 minutes to 1 hour. B: Overexpression of EGR-1 through Ad-Egr-1 in Beas-2B cells could significantly enhance GGPPS expression as CSE did. Total RNA and protein were collected and subjected to qPCR and Western blot analysis, respectively, after Ad-Egr-1 had been infected for 48 hours. C: The efficiency of EGR1 siRNA in Beas-2B cells. Beas-2B cells were transfected with EGR1 siRNA for 48 hours. Total RNA and protein were extracted and subjected to qPCR and Western blotting, respectively. D and E: Beas-2B cells were transfected with EGR1 siRNA or a scrambled control (D) and infected with DnEGR-1 adenovirus or a control adenovirus (Ade) (E) for 48 hours. Each group was then treated (10% CSE) or not treated (CTL) with CSE for 4 hours. Total RNA was collected and subjected to qPCR. The overexpression efficiency of DnEGR-1 is shown in panel E by Western blot. F: Beas-2B cells were transfected with DnEGR-1 and the luciferase reporter that contained the wild-type GGPPS promoter. The Renilla luciferase plasmid was used as an internal control. After 10% CSE stimulation for 20 hours, luciferase activity was determined. The data were normalized by the Renilla luciferase activity and expressed as a fold-induction with respect to control cells. G: Beas-2B cells were transfected with the luciferase reporter that contained the wild-type or mutant GGPPS promoter. The Renilla luciferase plasmid was used as an internal control. After 10% CSE stimulation for 20 hours, luciferase activity was determined. The data were normalized by the Renilla luciferase activity and expressed as a fold induction with respect to control cells. (*P < 0.05: compared with the control group untreated with CSE; †P < 0.05: compared with CSE treatment only; ‡P < 0.05: compared with the Ade group; and §P < 0.01: compared with the scrambled group).