KDM3A is a negative regulator of ciliogenesis. (A) Total cell extracts of wild-type (RPE1^WT) and KDM3A gene-edited RPE1 mutants (KDM3A^Cr22, named herein KDM3A^null) immunoblotted with KDM3A, revealing KDM3A-null clones (1, 2, and 8). (B) Mean ± 5th–95th percentile of ciliary lengths (N = 4) of wild-type lines (RPE1^WT, RPE1^CrWT) and the KDM3A^null lines (1,2,8) after 3 h or 24 h without serum (−FCS) and after 24 h −FCS followed by 15-min temperature stress (42°C) in the presence of serum or 90-min serum only. Dots represent cilial lengths outside the indicated percentile (n > 100 cilia per experiment). One-way analysis of variance, ****, P < 0.0001; paired t test, **, P < 0.01; ***, P < 0.001; ns, not significant. The F-test illustrates the length variance between RPE1^WTs and KDM3A^nulls for each ciliary phase. (C) Percentages of ciliated cells after 24 h −FCS at the indicated times after serum replenishment. (D) Segmented nuclei and cilia (stained with anti-ARL13B) of RPE1^CrWT and KDM3A^null (line 1) cultures after 3-h serum depletion highlights differences in the frequency of ciliated cells (41%, SD = 7.6; 64%, SD = 7.02, respectively), in spite of identical cell densities. Percentages are from n > 100 cells per experiment done in triplicate. χ² < 0.001. (E) Wide-field images illustrating the extreme range of lengths often observed in exponentially growing KDM3A^null lines (24 h FCS), with frequent abnormal ciliary bulges in mutants (arrow). (F) Percentage of cycling cells (24 h +FCS) that contain a cilium in control (RPE1^WT and RPE1^CrWT) and KDM3A^null (lines 2 and 8) cells (N = 3). ***, P = 0.001 is from χ² test. (G) Scoring of cilia found in late G2 (24 h +FCS or during serum induced ciliary resorption). WTs represent RPE1^WT and RPE1^CrWT, and KDM3A^null lines 1, 2, and 8. P-value is from χ² test. (H) Example of a mutant cilium remaining after centrosomes have separated (>2 µm). (I) Cell cycle profile of cycling (24 h +FCS) control (RPE1^WT and RPE1^CrWT) and KDM3A^null (lines 1, 2, and 8) cultures. Error bars represent SD.