Abemaciclib rescues Shh signaling in JBTS cells by relieving CDK6’s inhibition on axoneme polyglutamylation. (A) The knockdown efficiency of Armc9 siRNA in NIH-3T3 and IMCD3 cells was accessed by qPCR. Quantified data are presented as mean ± SD. Statistical analyses were performed by unpaired Student’s t test. N = 3 independent experiments. ***P < 0.001. (B and C) Knockdown of Armc9 or Cep41 does not affect ciliary base localization of CDK6 or FIP5 in NIH-3T3 cells. (D) Knockdown of Armc9 or Cep41 does not affect SAG induced ciliary translocation of SMO in NIH-3T3 cells. (E) The knockdown efficiency of mouse Cdk4 and Cdk6 specific siRNAs in NIH-3T3 cells were accessed by western blotting. The molecular weigth standards (kD) are labeled on right. (F and G) Knockdown of Cdk6, but not Cdk4, restores SAG-induced cilia tip accumulation of GLI3 in Armc9- (F) or Cep41- (G) depleted NIH-3T3 cells. (H) Abemaciclib does not affect axoneme acetylation or detyrosination. NIH-3T3 cells were transfected with siRNA for 48 h, followed by serum starvation and Abemaciclib (200 nM) treatment for 24 h. The mean fluorescent intensity of acetylated and detyrosinated tubulin (Detyr. Tub) in cilia was quantified. Quantified data for C–H are presented as mean ± SEM. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post-hoc test for multiple comparisons. N ≥ 40 cilia. *P < 0.05; **P < 0.01; ***P < 0.001. n.s: not significant. Scale bars: 2 μm.