Figure 7

(A) Real time PCR analysis show Chelerythrine-mediated repression of VEGFA, KRAS and BCL2 genes expression in MCF7 cells. Bar diagram showing relative expression level, quantified using real time PCR analysis, of VEGFA, KRAS and BCL2 genes after treatment of cells with 15 and 150 nM (n = 3 batches of cells for each treatment) Chelerythrine for 24 h. Control received only the buffer in which Chelerytrine was suspended. Data presented as relative expression level (±Standard Error) where expression in control calculated as almost 1 (±Standard Error). Expressions in different samples normalized based on expression of GAPDH gene. *significantly changed from control, and **significantly changed from cognate 15 nM sample (indicated with dashed line). (B–G) Chelerythrine downregulates the promoter activity of BCL2, KRAS and VEGFA through targeting G-quadruplex, confirmed by luciferase assay. (B,D,F) Schematic representations of the reporter luciferase constructs are given. BCL2 G-Quadruplex (GQ), KRAS GQ, and VEGFA GQ are the quadruplex- scaffolds which are located at the upstream of hRlucCP. GQ deleted constructs are also demonstrated below. (C,E,G) Promoter activity is shown in terms of relative luciferase units, which dictates the expression of the reporter gene. Significant reduction in luciferase activity is found for the constructs which harbour the quadruplex motifs in the upstream of promoters compared to those which are devoid of quadruplex motifs. Error bars in the bar plots (E,F and G) represent means ± s.d. from three independent experiments. Asterisks (*) indicate statistical significance as determined from Student’s t-test (*indicates P < 0.05, **indicates P < 0.01, ***indicates P < 0.001), which denote significant differences in the promoter activities of BCL2, KRAS, and VEGFA, compared with values for untreated cells (control).