Fig. 4

Effect of berberine on the promoter activity of the RET gene. a Effect of berberine on the luciferase expression in HEK293-WT and HEK293-MT1 cell lines following the treatment with berberine up to 24 h. Luciferase activity in cell lysates was measured as relative luminescence units (RLU) and normalized to the total protein content. Experiments were performed in triplicate. Error bar represent one s.d. above and below the mean % luciferase activity. b DMS foot printing on the RET-WT G-quadruplex forming sequence in the absence and in the presence of berberine (5 Equivalents) following 0.2 % DMS treatment (lanes C & B respectively). Purine & pyrimidine sequencing act as single base ladders to identify the protected and cleaved guanines after piperidine treatment (lanes 1 & 2 respectively). c Schematic models for the parallel G-quadruplexes formed by RET-WT (d) DMS foot printing on the RET-MT1 quadruplex forming sequence in the absence and in the presence of 5 equivalents of berberine following 0.2 % DMS treatment (lanes C & B respectively). e ChIP analysis to determine the effect of berberine in recruiting the SP1 and RNA pol II to the GC box region in the RET promoter region in TT cells following 48 h exposure at two different concentrations. Recruitment of SP1 and Pol II to the RET proximal promoter region was assessed by PCR using RET promoter specific primers. 1 % of the input DNA was used as internal control (Input) and isotype-matched IgG was used as a negative control for immunoprecipitation (Ig)