Fig. 4

Solamargine inhibited SP1 protein expression through inactivation of Akt; exogenous expression of SP1 abrogated the effect of solamargine on EP4 expression. a A549 and H1975 cells were treated with increased concentrations of solamargine for 24 h. Afterwards, the expression of SP1 proteins was detected by Western blot. b A549 and H1975 cells were transfected with the control or expression construct of Akt vectors for 24 h before exposing the cells to solamargine for an additional 24 h. Afterwards, the expression of Akt and SP1 proteins were determined by Western blot, and was expressed as percentage of control in the mean ± SD of three separate experiments. GAPDH was used as loading control. c A549 and H1975 cells were transfected with the control or expression constructs of SP1 for 24 h before exposing the cells to solamargine for an additional 24 h. Afterwards, the expression of SP1 and EP4 proteins were determined by Western Blot, and was expressed as percentage of control in the mean ± SD of three separate experiments. GAPDH was used as internal control. d A549 and H1975 cells were transfected with the control or expression construct of SP1 and wild type EP4 promoter reporter construct ligated to luciferase reporter gene and internal control for 24 h before exposing the cells to solamargine for an additional 24 h. Afterwards, the promoter activities were determined using the Dual-Luciferase Reporter kit (Promega). Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from the solamargine treated alone group (P < 0.01)