Figure 6. p27^CK– interferes with citron-K function.

(A) p27^CK– does not affect the multinucleation caused by citron-K depletion. HeLa cells were transfected with scrambled (scr) or citron-K siRNAs and plasmids expressing p27^CK– or empty vector (pQP). Citron-K knockdown was controlled by immunoblotting with goat anti-citron antibodies; β-tubulin levels were used as loading control. Multinucleation was quantified in cells (500 cells/condition, n = 3) stained with β-catenin and p27 antibodies and Hoechst (original magnification, ×400). Results were analyzed using 1-way ANOVA with the Tukey-Kramer multiple comparison test; *P < 0.05. (B) The minimal p27-binding domain of citron-K (HR1) rescues the multinucleation caused by p27^CK– overexpression. HeLa cells were transfected with p27^CK– and/or different domains of citron-K. Multinucleation was quantified (500 cells/condition, n = 3). Results were analyzed by 1-way ANOVA with the Newman-Keuls test; ***P < 0.001. (C) The HR1 domain rescues the multinucleation in p27^CK– primary MEFs. p27^CK– primary MEFs were transfected with GFP or with the HR1 domain, and multinucleation was evaluated 48 hours later (350–600 cells/condition, n = 3). Results were analyzed using Student’s t test; *P < 0.05. (D) RhoA depletion has no effect on p27^CK–-induced multinucleation. HeLa cells were transfected with scrambled or RhoA siRNA and plasmids expressing p27^CK– or empty vector (pQP). RhoA knockdown and p27 expression were controlled by immunoblotting with anti-RhoA and anti-p27 antibodies; β-tubulin levels were used as loading control. Multinucleation was quantified (500 cells/condition, n = 3). Results were analyzed as in A; *P < 0.05, **P < 0.01. (A–D) Error bars represent SEM.