CDK6, but not CDK4, suppresses axoneme polyglutamylation. (A) Ectopically expressed and endogenous CDK7 localize at the nucleus. (B) The knockdown efficiency of CDK4 and CDK6-specific siRNAs was accessed by western blotting. The molecular weigth standards (kD) are labeled on the right. (C) Knockdown of CDK6, but not CDK4, promotes axoneme polyglutamylation in RPE-1 cells. (D) The knockout validation of CDK4 and CDK6 by western blotting in RCTE cells. The molecular weigth standards (kD) are labeled on the right. (E) Knockout CDK6, but not CDK4, promotes axoneme polyglutamylation in RCTE cells. (F) The CDK6^−/− RCTE cells were transfected with indicated mCherry-tagged CDK6. 48 h later, cells were cultured in serum-free medium for 24 h to induce ciliogenesis. The length of polyglutamylated axoneme in mCherry-positive cells was measured. (G) The RPE-1 cells were transfected with mCherry-tagged CDK6^T177D. 48 h later, cells were cultured in a serum-free medium and treated with THZ1 for 24 h. The length of polyglutamylated axoneme was measured. (H) Abemaciclib (Abe) restores axoneme polyglutamylation in TTLL5-depleted RPE-1 cells. Cells were transfected with TTLL5 siRNA for 48 h, followed by Abemaciclib (500 nM) treatment for 24 h in serum-free medium. (I) Abemaciclib partially rescues the axoneme polyglutamylation in CCP5-YFP overexpressed RPE-1 cells. Quantified data are presented as mean ± SEM. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post-hoc test for multiple comparisons. ***P < 0.001. n.s: not significant. N ≥ 50 cilia. Scale bars: 5 μm (A) or 2 μm (C, E, H, and I). Source data are available for this figure: SourceData F2.