Fig. 3
KIF24 recruits MPP9 at the distal end of the mother centriole in hTERT RPE-1 cells. a Lysates from hTERT RPE-1 cells were subjected to immunoprecipitation (IP) with anti-MPP9 antibody, and immunoblotting with the indicated antibodies. b Immunoblots showing depletion of MPP9 or KIF24 by siRNA in hTERT RPE-1 cells. Tubulin was used as a loading control. c Immunostaining of acetylated-tubulin (Acet-Tub, red) and MPP9 (green) in the control- or KIF24-siRNA-treated hTERT RPE-1 cells. d Quantification of the percentage of cells with the indicated number of MPP9 dots from c. e Immunostaining of Acet-Tub (red) and KIF24 (green) in the control- or MPP9-siRNA-treated hTERT RPE-1 cells. f Quantification of the fluorescence intensity of KIF24 at centrosomes from e (n = 100 cells for each group). g Immunostaining of MPP9 (green) and Flag (red) in KIF24-3’UTR siRNA-treated or/and Flag-KIF24-rescued hTERT RPE-1 cells. h Immunoblots showing depletion of KIF24 by 3’UTR siRNA transfection or/and rescue of KIF24 expression by overexpression of Flag-KIF24. Tubulin was used as a loading control. i Quantification of the percentage of cells with 4-dot MPP9 in control-, KIF24-3’UTR siRNA, or/and Flag-KIF24-rescued hTERT RPE-1 cells. j Immunoblots showing depletion of MPP9 or/and KIF24 by siRNA in hTERT RPE-1 cells. Tubulin was used as a loading control. k Quantification of ciliogenesis in control-, MPP9-, or/and KIF24-siRNA-treated hTERT RPE-1 cells. l Immunoblots showing depletion of MPP9 or KIF24 by siRNA in U2OS cells. Tubulin was used as a loading control. m Quantification of the percentage of cells with the indicated number of MPP9 dots in control- and KIF24-siRNA-treated U2OS cells. For d, f, i, k, and m, bars represent the means ± S.E.M for three independent experiments. n.s., not significant, *p < 0.05, **p < 0.01, as determined by unpaired two-tailed Student’s t-test (d, f, k, m) and one-way ANOVA analysis (i). Scale bars: 1 μm (c, e, g)
