EMX2 expression in U87 transfected cells. a- Production of a tetracycline-regulated EMX2 expression system in U87 cells. Experimental design. Six distinct, stable, double transfected clones were constructed. First, U87 cells were transfected using the regulatory vector pcDNA6/TR. The two resulting clones (TR cl. A and TR cl. B) were further transfected by means of the expression vector pcDNA4/TO/myc-HisA_EMX2. We selected three stable clones derived from each regulator clone, TR cl.A (EMX2 cl.A.1, EMX2 cl.A.2 and EMX2 cl.A.3) and TR cl.B (EMX2 cl.B.1, EMX2 cl.B.2 and EMX2 cl.B.3). b- Phenotype induced by EMX2 restoration. 16 days cell culture with or without EMX2 induction were carried out for the 6 clones described in A. This design allowed testing for (1) the EMX2 overexpression phenotype (Tet); (2) the reversibility of this phenotype by Tet-induction arrest at day 8 (D8 Tet). Control conditions correspond to culture without tetracycline (No Tet). The same experiments were also performed using empty plasmid clones as described in Material and Methods. Complete sets of clones and associated conditions are depicted in Table A (Supplementary data) c-d- EMX2 expression in the tetracycline-inducible system. EMX2 mRNA levels in distinct clones: six independent clones were used (the three clones derived from the regulator clone TR cl.A and the three clones derived from the TR cl.B). EMX2 mRNA level was measured at day 0 (no induction), day 2 and day 6 after tetracycline-induction (c). Welch Two Sample t-test on EMX2 cl.A. (J2 versus no Tet p = 0,00375; J6 versus no Tet p = 0,00008) and on EMX2 cl.B. (J2 versus no Tet p = 0,00161; J6 versus no Tet p = 0,00091). EMX2 protein levels after six days of culture with (+) or without (−) tetracycline-induction in empty clones (Control) and two independant EMX2 clones (d).