CK2 activity in GL261 cells and tumour samples. (A) Western blot for GL261 cell protein extracts (25 µg) treated with increasing doses of CX-4945 (from left to right: control (C) and CX-4945 treated cells 5 µM, 10 µM, 20 µM, 30 µM and 60 µM). This experiment was performed with n = 1 for each condition and for 8 h (upper part) or 24 h (lower part). p-Akt(S129), Akt1 total and β-Tubulin proteins were analysed; (B) Western blot for GL261 cell protein extracts (25 µg) treated with 67.2 µM CX-4945 (from left to right: control (C) and CX-4945 treated cells for 1 h, 4 h, 8 h, 12 h and 24 h). The experiment was performed with n = 3 for each condition. p-Akt(S129), Akt1 total, CK2α, CK2β and β-Actin proteins were analysed; (C) Quantification of western blot (WB) for GL261 cell protein extracts (25 µg) treated with CX-4945 (n = 3 per each condition). Ratio (%) of p-Akt(S129) content divided by Akt1 total content, while the control values (C) for this ratio are taken as the 100% start value. * = p < 0.05 for Student’s t-test for the comparison of control and treated groups; (D) CK2 activity measured on a CK2-specific synthetic peptide in lysates from GL261 cells treated with 67.2 µM CX-4945 (n = 3 for each condition). Treatment during 1 h, 4 h, 8 h, 12 h and 24 h. * = p < 0.05 for Student’s t-test for the comparison of control (100% initial value) and treated groups; (E) Boxplot of CK2 activity in CX-4945 treated mice compared to control mice. CX-4945 was administered to treated mice during 3 days (a total of 150 mg/Kg/day split into two administrations per day) and mice were euthanized 2 h, 6 h and 24 h after the last CX-4945 administration. As no CK2 activity differences (p > 0.05) were detected between euthanization time points (2 h, 6 h and 24 h), they were grouped in a single CX-4945 treated group. (* = p > 0.05 for Student’s t-test for the comparison of control and treated groups). CK2 activity was measured on tissue homogenates by means of radioactive assays towards a CK2-specific peptide. Boxplot features as in Figure 1 legend.