Figure 2

Luciferase reporter activation by single site variants. (A) HaCaT cells were co-transfected in triplicate with GLI2act expression constructs and the PTCH1_VAR luciferase reporter containing the consensus or a single site variant or the 6G7G (negative control). LacZ expression plasmid was added for normalization to all transfections. RLU for each transfection were first normalized with respect to lacZ expression and then with respect to the mean of RLU of three consensus samples from the same 24 well plate. The boxplot represents the results of two independent experiments normalized separately and then pooled to result in 6 measurements per position and base. Boxplots indicate the range (whiskers), estimated lower and upper quartile (box) and median (line) of the normalized values, which are expressed as percent of consensus activity. The consensus sequence in each position is coloured red and the corresponding base marked with an asterisk. A statistical analysis using an exact Kruskal-Wallis test rejects the null-hypothesis of equal population medians at significance levels p < 0.01% (that is, highly significant) for all positions with the exception of position 5, for which p = 46%. (B) Wilcoxon rank sum tests were done comparing the values of the base in each position to the negative control 6G7G (left panel) and to the consensus site (right panel). On the left, the consensus is highlighted by asterisks, on the right, the consensus positions have been left empty. p-values below 0.01 (1%) indicate clear rejections of the null-hypothesis of the medians of the distributions being equal. There are only 4 non significant values on the left, among them no single consensus. The right panel supports the boxplots in (A). Note, that due to the small sample size, only few numerical values are possible in all the (exact) Wilcoxon tests.