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Product Usage Information

CST recommends transfection with 100 nM p16 INK4A siRNA I 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use.

Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 μl per well.

Storage

SignalSilence? siRNA is supplied in RNAse-free water. Aliquot and store at -20oC.

Background

Cyclin-dependent kinases (CDKs) are activated in part by forming complexes with cyclins. For example, CDK4 and CDK6 associate with the D-type cyclins and phosphorylate the retinoblastoma protein. This phosphorylation is a necessary event for cells to enter S-phase (1). The inhibitors of CDK4 (INK4) family include p15 INK4B, p16 INK4A, p18 INK4C and p19 INK4D. p18 has been shown to function as a haploinsufficient tumor suppressor in vivo (2). All INK4 proteins are composed of 32 amino acid ankyrin motifs and selectively inhibit CDK4/6 activity. Mutational analyses of p18 implicate the third and the amino-terminal portion of the fourth ankyrin repeat in mediating binding to CDK4/6 (3). The interaction of INK4 family members can be a binary complex with CDK4/6 or ternary complex with cyclin D-bound CDK4/6 and ultimately results in the inhibition of cell cycle progression (4,5).

p16 INK4A directly inhibits the activity of cyclin D, thereby inhibiting S-phase entry (6,7). As such, expression of p16 INK4A is commonly associated with cellular senescence, and disruption of the p16 INK4A gene is frequently observed in human cancers.

1. Lukas, J. et al. (1996) Mol Cell Biol 16, 6917-25.
2. Bai, F. et al. (2003) Mol. Cell. Biol. 23, 1269-1277.
3. Noh, S.J. et al. (1999) Cancer Res. 59, 558-564.
4. Guan, K.L. et al. (1994) Genes Dev 8, 2939-52.
5. Hirai, H. et al. (1995) Mol Cell Biol 15, 2672-81.
6. Sherr, C.J. (2001) Nat Rev Mol Cell Biol 2, 731-7.
7. Lowe, S.W. and Sherr, C.J. (2003) Curr Opin Genet Dev 13, 77-83.