Perturbing actin polymerization phenocopies IFT abnormalities of KDM3A mutants. (A) Single time frames from Videos 1 and 2 of live RPE1^WT and KDM3A^null (line 2) cultures. Arrows point to bulges forming along KDM3A^null cilia. (B) Confocal images after 48 h −FCS indicate low levels of IFT81 at tip of RPE1^WT cilia (green arrow) but conspicuous accumulations in KDM3A^null mutant cilia, frequently accompanied by IFT81 containing “vesicles” (yellow arrows). (C) Wide-field images of RPE1^WT cultures in the presence of serum show accumulations of endogenous IFT81 in CytoD treated cells that appear sequentially as ciliary buds (cb; without acetylated α-tubulin–stained axonemes, most frequent with 0.2 µM CytoD) and progress toward ciliary tip (ct; 2 µM CytoD). (D) Representative images of IFT81 distribution in cilia formed after 3 h with solvent control or 100 µM CK-869 −FCS. (E) Quantification of cilia with or without IFT81 bulges among treated (2 µM CytoD and 100 µM CK-869) and solvent controls. Numbers in bars indicate number of cilia scored. Error bar represents SD. P-value is from χ² test. (F) Frequency of length ranges illustrate increased proportion of long cilia in 100 µM CK-869–treated cultures. (G) Differential sensitivity of RPE1^WT and KDM3A^null to ARP2/3 inhibition as illustrated by the length of cilia in subconfluent cultures (50–70 cells/0.1 mm²) treated for 3 h with 2 and 10 µM CK-869 −FCS. Mean ± 5th–95th percentile (N = 3 replicates, n > 30 cilia measured/condition). (H) Mean ± 5th–95th percentile of ciliary lengths indicate that RPE1^WT cultures begin to show significant cilial elongation in ≥50 µM CK-869 (N = 3 replicates, n > 30 cilia measured/condition). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (paired t test); ns, not significant. Bars, 5 µm.