Fig 1: CLK2 regulates the phosphorylation of AKT and FOXO3a proteinA, the graph shows that the phosphorylation for AKT, FOXO3a, and mTOR normalized total expression of AKT, FOXO3a, mTOR in vector- and CLK2 shRNA-infected GSC272 cells. The gene expression levels were determined using RPPA analysis. B and C, GSCs of vector and CLK2 knockdown were incubated with bFGF and EGF for 1 hour. The cells were harvested, lysed, and analyzed using Western blotting. D and E, GSCs were incubated with bFGF and EGF for 24 hours. Phosphorylation of FOXO3a was assessed using Western blotting D, and immunofluorescence E. Phosphorylated FOXO3a stained red, and the nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue).
Fig 2: Knockdown of CLK2 expression arrests the cell cycle at G1 and S phase and increases p27 expression in GSCsA, GSCs were incubated with 70% EtOH at -20°C for 1 day and washed three times with PBS and a propidium iodide staining solution. B, GSCs were incubated with bFGF and EGF in B27 media for 3 days, and single cells were treated with Accutase. The cells were stained with annexin V and 7-AAD, and apoptosis of the cells was detected using FACS analysis. C, after knockdown of CLK2 expression, expression of p27 protein in several GSC lines was measured using Western blotting.
Fig 3: Expression of CLK2 in human glioblastoma specimens is inversely correlated with patient outcomeA, immunohistochemical analysis of CLK2 expression in human glioblastoma specimens. CLK2 expression was independently validated using a glioblastoma tissue microarray. Representative immunohistochemical images of overall CLK2 expression in glioblastoma tissue microarray are shown (magnification, x100). Tissue punches were scored for CLK2 expression as 0 (negative/absent expression), 1 (weak expression), 2 (moderate expression), or 3 (strong expression). B, Kaplan-Meier survival analysis according to CLK2 expression by immunohistochemistry and log rank test was used to test for significant differences. C, immunohistochemical analysis of FOXO3a phosphorylation in the glioblastoma tissue microarray. Phosphorylation of FOXO3a was positively correlated with CLK2 expression.
Fig 4: Role of CLK2 expression in GSCsA, GSC lysates were collected, and CLK2 expression in the lysates was detected using Western blot analysis. B, GSC11, GSC272, GSC7-2 and GSC17 cells were infected with a lentivirus containing a control vector, CLK2 shRNA1, or CLK2 shRNA2. C, Hierarchical clustering was applied to protein expression data from RPPA results in vector shRNA and CLK2 shRNA in GSC272 cells. The protein expression levels in CLK2 shRNA-infected cells differed at least 1.6-fold from those in control GSC272 were selected for hierarchical clustering analysis. The red and green colors in the cells reflect relatively high and low expression, respectively. D, the cells were infected with a lentivirus containing a control vector or CLK2 shRNA, a volume of CellTiter-Glo Reagent was added to cells, and the luminescence was recorded. E, the GSCs per well were added to a 96-well plate with 20 µl of a combined MTS/PMS solution, and the plate was incubated for 3h 30min at 37°C in a humidified atmosphere. *P < 0.05; **P < 0.01; ***P < 0.001 versus control.
Fig 5: Depletion of CLK2 decreases the expression of Ki-67 and phosphorylation of AKT/FOXO3aA-B. immunohistochemical stains of GSC11 and GSC272 tumor samples obtained from orthotopic murine xenografts. A, immunofluorescent stains for CLK2 and the stem cell marker nestin in GSC11 and GSC272 mouse xenografts with or without infection with CLK2 shRNA are shown. CLK2 was labeled as red and nestin as green. Expression of CLK2 and nestin overlap. B, Ki-67–positive cells stained brown with hematoxylin counterstain of nuclei. The bar graph shows the numbers of Ki-67–positive cells in vector-infected and CLK2-knockdown xenografts. C, immunofluorescent stains for phosphorylated AKT and FOXO3a in murine GSC11 and GSC272 cells. Phosphorylated AKT and FOXO3a stained red, and the nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue).
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