Usp14 controls ciliogenesis. (A) Validation by WB of Usp14 depletion in MEFs transfected with siRNAs targeting Usp14 (Usp14 siRNA) and non-targeting negative control siRNAs (Control siRNA). (B) Representative confocal images of primary cilia in cycling MEFs in complete medium (with FBS), transfected with Usp14 siRNA and control siRNA for 96 h. Acetylated tubulin (red) marks cilia. Hoechst (blue) labels nuclei. Histogram shows the quantification of percentage of ciliated cells in this culture condition. (C) Representative confocal images of primary cilia in cycling MEFs in complete medium, treated with IU1 or DMSO for 0, 6 and 24 h. Acetylated tubulin (red) marks cilia. Hoechst (blue) labels nuclei. Histogram, bottom left, shows the quantification of percentage of ciliated cells. (D) Analysis by WB to verify suppression of ubiquitin chain trimming by IU1 at 0, 6 and 24 h of treatment. The bands are at the same exposure, non-contiguous on the same gel. Data are expressed as mean ± SEM (n = 3 independent experiments); *P < 0.05; **P < 0.01; ***P < 0.001, one-tailed Student’s t-test (A) and the likelihood ratio test for negative binomial generalized linear models (B and C) were applied; ≥250 cells (B) and ≥140 cells (C) were analyzed per sample; Scale bar = 5 μm.