CDKN2A negatively regulated pRb and down-regulated level of cell cycle regulatory protein cyclin D1. Western blot analysis revealed a markedly lower phosphorylation of pRb and expression of cyclin D1 in T98G, U87-MG and SW1783 glioma cell lines transfected with CDKN2A (A). However, knockdown of CDKN2A increased the phosphorylation of pRb and cyclin D1 in H4 glioma cell line. Moreover, a cyclin D1 inhibitor flavopiridol blocked the elevated phosphorylation of pRb and the expression of cyclin D1 induced by CDKN2A knockdown (B). Increased cyclin D1 also detected in high-grade gliomas tissues comparing low-grade gliomas tissues (C). Three independent experiments were preformed. A representative result was shown. pRb, phosphorylated Rb; tRb, total Rb. Actin as a loading control.