Fig. 6

Functional effects of the overexpression of EMX1 and EMX2 in combination with constitutive activation of the Wnt pathway on tumorsphere (TO) formation. A-L Tumorsphere (TO) formation assay results for models of overexpression of EMX1 (E1) or EMX2 (E2) and CTNNB1 (β-CAT) in AA cells (A, B and C) and AW cells (G, H and I). Models of joint overexpression of EMX1, EMX2 and CTNNB1 in AA cells (D, E and F) and AW cells (J, K and L). In A, D, G and H the mean number of tumorspheres generated by AA cells (A and D) or AW cells (G and J) is represented. The mean sizes of the tumorspheres generated by AA cells (B and E) and AW cells (H and K). In C, F, I and L representative micrographs of the tumorsphere formation results for AA cells (C and F) and AW cells (I and L). The mean of a minimum of 3 independent experiments performed in triplicate ± standard deviation are shown. TO size is given as μM diameter. M–O Effects of EMX1 and/or EMX2 overexpression in combination with constitutive activation of the Wnt pathway on genes of the canonical Wnt pathway. M, Ñ Quantification of the relative mRNA levels of the AXIN1, GSK3β, TCF4, WNT1, and CCND1 determined by qRT-qPCR in AA cells (M) and AW cells (Ñ) overexpressing EMX1 or EMX2 and CTNNB1. N and O Quantification of the relative mRNA levels of the same genes in AA cells (N) and AW cells (O) jointly overexpressing EMX1, EMX2 and CTNNB1. Graphs of the expression levels (2-ΔCt) for total extracts of each cell line are shown. The mean of a minimum of 3 independent experiments performed in triplicate ± standard deviation are shown. Statistical analysis was performed with Student's t test (* p < 0.05; ** p < 0.01; *** p < 0.001)