KDM3A regulates the expression of and interacts with actin components. (A) Schematic of experiments used to isolate RNA and protein lysates during ciliary growth and resorption. (B) Phalloidin staining and histograms of pixel intensities reveal decreased abundance of stress fibers in KDM3A^null cells. (C) Principal-component analysis of total cell proteomes from RPE1^WT and KDM3A^null cells during ciliary growth and resorption show that variability between growth conditions (43.8%) is larger than that between genotypes (15.7%). (D) Venn diagram illustrates the number of significantly down-regulated genes in KDM3A^null compared with RPE1^WT in the transcriptomic and proteomic datasets during ciliary growth and resorption. Dysregulation of extracellular environment is the most enriched term by GO analysis (Table S4). (E) Label-free quantification (LFQ) intensities for peptides specific to ACTA2 and ACTG2 (nonseperable because of sequence homology) across the indicated growth conditions obtained from total cell proteomes of control (RPE1^WT and RPE1^CrWT) and KDM3A^null (line 2) lines. **, P < 0.001; ns, not significant. (F) Proteomic-based identification of endogenous components significantly enriched in KDM3A pull-downs during ciliary growth (3 h −FCS). P-values are from t tests of label-free quantifications from RPE1^WT relative to KDM3A^null from three experimental replicates. Select isolated actin-binding proteins are highlighted. (G) Schematic of the KDM3A region used as bait (JMJC domain in red) and ACTA2 prey identified in the two-hybrid screen. (H) Independent colonies (one to three) of rescued bait/prey plasmid cotransformed onto two-hybrid Gold yeast and grown in double selection (Leu/Trp) were resuspended and assessed for aureobasidin A resistance and α-Gal activity. Only yeast cotransformed with JmjC and ACTA2 grew in the stringent conditions indicating a binary interaction. (I) Pure smooth muscle actin resolved in 4–12% acrylamide gel (three serial dilutions) transferred to nitrocellulose is bound by GST-JMJC, but not GST.