Fig. 6.

Oxidation of p16^INK4A impairs its inhibitory function towards CDK4/6.

(A) In vitro CDK4/6 kinase assay on WT p16^INK4A pulldowns shows that the oxidation of p16^INK4A-C72 impairs its inhibitory function. Note that oxidation or mutation of Cys72 does not affect the amount of CDK4/6 that is co-immunoprecipitated. (R: Reducing, NR: Non-reducing). All Western blots shown in Fig. 6 are typical results of several repeats (N ≥ 3 for all experiments).

(B) qPCR analysis showing the expression of WT p16^INK4AWT or p16^INK4A-C72A (top) in COLO-829 cells upon induction of expression from the respective pINDUCER20-p16^INK4A construct. CCNE2 expression (bottom) is strongly repressed both by WT p16^INK4A and p16^INK4A-C72A. Diamide treatment relieves this repression only in the WT p16^INK4 expressing cell line. Note that the inducible system is somewhat leaky, which could explain induction of CCNE2 in the p16^INK4AWT cells by diamide in the absence of doxycycline. The mean plus SEM is displayed from n = 5 biological replicates. *p < 0.05, **p < 0.01, pairwise comparison using a two-sided t-test comparing cells carrying the inducible WT p16^INK4 or p16^INK4A-C72A construct in each condition.