Loss of CDKN3 expression and elevated CDK activity in glioblastoma. (A) CDKN3 is ubiquitously expressed in human brain. (B) The CDKN3 protein is expressed in healthy brain (red arrows). CDKN3 expression is diminished (green arrows) in human GBM tumors. Images are shown at 100× magnification. (C) Immunohistochemistry quantification confirms loss of the CDKN3 protein in GBM. Percentages of CDKN3-positive cells in healthy brain cores and tumor specimens were compared by t test (P = 0.0003). Error bars represent mean values ± SEM. (D) Activation of CDKs in GBMs. Immunohistochemistry reveals increased CDK substrate phosphorylation in brain tumors compared with normal brain. (E) Quantification of increased CDK activity in GBMs. Percentages of phospho-CDK substrate–positive cells in healthy brain cores and tumors were compared by paired t test (P = 0.0133). Error bars represent mean values ± SEM. (F) Decreased expression of CDKN3 accompanied by CDK activation in an independent cohort of human GBMs. Note the increased phosphorylation of CDK substrates in all tumors. (G) Quantification of CDKN3 expression in healthy brain and GBM. CDKN3 and actin were quantified in at least three independent Westerns per specimen, and the ratios were compared with healthy brain (one-way ANOVA; n = 3 experiments for each tumor and 13 experiments for healthy brain). Yellow columns indicate tumors with significantly decreased total CDKN3 expression. Broken lines: 95% confidence interval. Error bars represent mean values ± SEM. (H) Quantification of the CDKN3 expression in 26 GBMs. 23% of tumors (6/26) showed significant loss of CDKN3 expression (one-way ANOVA; n = 3 experiments for each GBM and 13 experiments for healthy brain).