Fig 2: CK2 inhibition reduces NG2 gene expression in human GBM cell lines. (A) A1207 wild type and CK2a KO cells were transfected with pGL4-NG2p1.2.4.1, cultivated for 24 h, lysed and the transcriptional activity was detected by a luciferase assay. Relative luciferase units (RLU) of wild type cells were used as control. Mean ± SD. * p < 0.05 vs. wild type (n = 4). (B) A1207 cells were transfected with pGL4-NG2p1.2.4.1 for 24 h, treated with vehicle (DMSO) or CX-4945 (10 µM) and analyzed by a luciferase assay. RLU of vehicle-treated cells were used as control. Mean ± SD. * p < 0.05 vs. vehicle (n = 3). (C,D) A1207 and U87 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h, harvested and total RNA was isolated. The relative gene expression of NG2 was examined by qRT-PCR normalized to GAPDH. Vehicle-treated cells were set 100%. Mean ± SD. * p < 0.05 vs. vehicle (n = 4). (E,F) A1207 and U87 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) in the presence of cycloheximide. The cells were scratched after 0 h, 24 h, 48 h and 72 h and the MFI of NG2-positive cells was assessed by flow cytometry. MFI of cells at 0 h was set 100%. Mean ± SD (n = 4).

Fig 3: CSPG4 expression in patient-derived matched pairs of melanoma tumor samples before and after therapy with kinase inhibitors. Tissue slides were prepared from biopsies of subcutaneous metastases of a melanoma patient before treatment (left panel) and after progression under therapy with dabrafenib and trametinib (right panel). (A and C) H&E staining, magnification ×100. (B and D) CSPG4 staining with anti-CSPG4 antibodies clone 132.38, magnification ×100. CSPG4, chondroitin sulfate proteoglycan 4; H&E, hematoxylin and eosin.

Fig 4: Effect of CK2 inhibition on NG2 expression. (A) Pericytes were treated with vehicle (DMSO), CX-4945 (10 µM) or TBB (50 µM) for 48 h. The cells were scratched and the mean fluorescence intensity (MFI) of NG2-positive cells was assessed by flow cytometry. Vehicle-treated cells were set 100%. Mean ± SD. * p < 0.05 versus vehicle (n = 4). (B–D) Pericytes were treated as described in (A). The cells were lysed and the expression of NG2, Akt, pAkt and a-tubulin (as loading control) was analyzed by Western blot (B). NG2/a-tubulin (C) and pAkt/Akt (D) were assessed by quantitative analysis of Western blots. Vehicle-treated cells were set at 100%. Mean ± SD. * p < 0.05 versus Vehicle (n = 4). (E–H) Pericytes were treated with a combination of CK2a and CK2a’ siRNA or with ctrl siRNA. The cells were lysed and the expression of NG2, Akt, pAkt, CK2a, CK2a’ and a-tubulin (as loading control) was analyzed by Western blot (E). CK2a/a-tubulin was assessed by quantitative analysis of Western blots (F). pAkt/Akt was assessed by quantitative analysis of Western blots (G). MFI of NG2-positive cells was detected by flow cytometry (H). ctrl siRNA-treated cells were set as 100%. Mean ± SD. * p < 0.05 versus ctrl siRNA (n = 4). (I) Pericytes were treated with vehicle (DMSO), CX-4945 (10 µM) or TBB (50 µM) in the presence of Cycloheximide (CHX) for 0, 24 and 48 h. Then, the cells were scratched and the MFI of NG2-positive cells was assessed by flow cytometry. Cells at 0 h were set 100%. Mean ± SD (n = 4). (J) Pericytes were transfected with ctrl-plasmid or NG2-plasmid for 24 h following treatment with vehicle or CX-4945. The cells were lysed and the expression of NG2 and a-tubulin (as loading control) was analyzed by Western blot. (K) Pericytes were treated as described in (A) and total RNA was isolated. The relative gene expression of NG2 was examined by qRT-PCR normalized to GAPDH as housekeeping gene. Vehicle-treated cells were set 100%. Mean ± SD. * p < 0.05 versus vehicle (n = 4). (L and M) Pericytes were treated as described in (A) and the MFI of ß1-integrin (L) and activated ß1-integrin (M) was assessed by flow cytometry. Vehicle-treated cells were set as 100%. Mean ± SD (n = 4).

Fig 5: CK2 inhibition reduces the NG2 expression of the JA cells. (A–D) The JA1 and JA2 cells were treated with CX-4945 (10 µM) and SGC-CK2-1 (5 µM) for 48 h. Vehicle-treated cells served as controls. The cells were lysed and the expression of NG2, CK2a, CK2ß and a-tubulin (as a loading control) was assessed by Western blot. The relative density ratio of NG2/a-tubulin (B,C), CK2a/a-tubulin (D,E) and CK2ß/a-tubulin (F,G) was determined and the vehicle-treated cells were set at 100%. Mean ± SD (n = 3). * p < 0.05 vs. vehicle. (H,I). The gene expression of NG2 was measured by qRT-PCR. Vehicle-treated cells were set at 100%. Mean ± SD (n = 3). * p < 0.05 vs. vehicle.