Implication of known ciliogenesis effectors in centrosome migration. (A) RPE1 EGFP-centrin1 cells were treated with siRNAs targeting known primary ciliogenesis effectors for 24 h to investigate their potential role in centrosome migration during primary cilium formation. The proportion of cells displaying a centrosome located >3 µm above the basal pole was determined and normalized to that of the nontargeting control siRNA for each condition. See Table S1 for the siRNA sequences and Fig. S1 (A and B) for the effect on the rate of ciliated cells and the length of primary cilia. (B) Side views of serum-starved RPE1 EGFP-centrin1 cells stained with DAPI to label the DNA and an antibody to α-tubulin to stain microtubules. Bars: (x and y) 10 µm; (z) 2.5 µm. (C) Staining of RPE1 cells with DAPI and with antibodies to acetylated tubulin and γ-tubulin. Images show maximal projection of z stacks. Bars: (top) 10 µm; (bottom) 1 µm. (D) Representation of synchronized time-lapse centrosome movement in serum-starved RPE1 EGFP-centrin1 treated with nontargeting control siRNA (one experiment, n = 32 cells) or siRNA against siCep164 (one experiment, n = 25 cells; left and middle). The graph represents the maximal centrosome z position for cells treated with nontargeting control siRNA and with siRNA against siCep164 (right). ****, P < 0.0001. Error bars represent standard deviation. Horizontal bars show mean values.