EMX2 induces a reversible proliferation block in U87 GB transfected cells. a- Cell proliferation was measured in three different culture conditions over 28 days without tetracycline (No Tet-black line), with tetracycline (Tet-dotted line) and with tetracycline for the first 8 days only (D8 Tet-gray line). Distinct triplicate experimental cultures were performed for empty vector and for all EMX2 cl.A or EMX2 cl.B (see Fig. 2). Proliferation curves were statistically validated using a linear mix model (see Materials and Methods)
b-c- Cell death was assessed before (time 0), 3 and 7 days after addition of tetracycline for empty clones (Control) and EMX2 cl.A clones (EMX2). After treatment, cells were stained with AnnexinV-PE and 7-AAD then analyzed by a FACscan flow cytometer. The labeling patterns identify different cell populations: vital cells (7AAD-negative/ annexin V-negative) in region Q3, apoptotic cells (7AAD-negative/annexinV-positive) in region Q4, dead cells (7AAD-positive/annexin V-positive) in region Q2 and damaged cells (7AAD-positive/annexin V-negative) in region Q1. Cell percentages are shown for Q3, Q4 and Q1 + Q2 regions (b). A representative histogram for viability, apoptosis and necrosis is shown in (c). Data are expressed as mean ± SE. Experiment was carried out in triplicates using three distinct clones. p value is not significant compared to control group. Effect of EMX2 overexpression on apoptosis in U87 GB cells (d). Caspase-3 activity was measured in cell lysates after 1, 3 and 7 days after Tet induction. Each column represents the mean value (+ SD) of three independent clones (n = 3, Student’s test) normalized to non-treated cells (taken as 100%). Positive controls were performed at each time point (0, 1, 3 and 7 days) and consisted of adding staurosporine (10 μM) 24 h before Caspase-3 activity dosage normalized to non-treated cells (taken as 100%) (n = 12, Student’s test, * p < 0.001).