Figure 7.
CCDC15 depletion results in reduced ciliation and inefficient redistribution of signaling proteins in response to Hedgehog stimulus. (A) Representative immunofluorescence images of ciliogenesis defects in RPE1 cells. Cells were transfected with control or CCDC15 siRNA. 72 h after transfection, cells were serum-starved for 24 h, fixed, and immunostained for the primary cilium with acetylated tubulin antibody (Acet-tubulin) and the centrosome with γ-tubulin antibody. DNA was visualized with DAPI. Yellow dashed boxeds mark the zoomed-in primary cilium. Scale bar, 10 μm, insets, 2 μm. (B and C) Quantification of ciliogenesis efficiency and length for A. Error bars, SD. n > 100 cells per experiment. Data represent mean value from three experiments per condition. Ciliation percentage: siControl = 70% ± 6, siCCDC15 = 46% ± 0.8, P = 0.0027. Cilium length: siControl = 3 μm ± 0.7, siCCDC15 = 2.3 μm ± 0.4, P < 0.0001, two-sided t test. (D) Representative U-ExM images of control and CCDC15-depleted RPE1 cells serum starved for 24 h. Centrioles and primary cilium were stained with tubulin (magenta) antibody and CCDC15 (green). Different ciliary defects associated with basal bodies efficiently depleted for CCDC15 were represented (panel on the right). Scale bar, 200 nm. (E) Representative immunofluorescence images of the effect of CCDC15 depletion on ciliary recruitment of SMO. Control and CCDC15 siRNA–depleted RPE1 cells were treated with 200 nM SAG for 24 h, fixed and stained for SMO, acetylated tubulin (Ac-tub), and DAPI. Yellow dashed boxes mark the zoomed-in primary cilium. Scale bar, 10 μm, insets, 1 μm. (F) Quantification of E. Error bars, SD. n > 50 cells per experiment. Data represent mean value from three independent experiments. SMO-positive cilia: siControl 56% ± 1, siCCDC15 25% ± 6, P = 0.0003, two-sided t test. (G) Representative images of the effect of CCDC15 depletion on basal body levels of IFT88. RPE1 cells were transfected with control or CCDC15 siRNA, fixed and stained for IFT88, acetylated tubulin, and γ-tubulin. DNA was visualized with DAPI. Yellow dashed boxes mark the zoomed-in primary cilium. Scale bar, 10 μm, insets, 1 μm. (H) Quantification of G. Graphs indicate IFT88 levels normalized to γ-tubulin levels at the basal body. Error bars, SD. n > 50 cells per experiment. Data represent the mean of three independent experiments. siControl = 1 ± 0.34, siCCDC15 = 0.67 ± 0.37. P < 0.0001, two-sided t test. ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: non-significant.

CCDC15 depletion results in reduced ciliation and inefficient redistribution of signaling proteins in response to Hedgehog stimulus. (A) Representative immunofluorescence images of ciliogenesis defects in RPE1 cells. Cells were transfected with control or CCDC15 siRNA. 72 h after transfection, cells were serum-starved for 24 h, fixed, and immunostained for the primary cilium with acetylated tubulin antibody (Acet-tubulin) and the centrosome with γ-tubulin antibody. DNA was visualized with DAPI. Yellow dashed boxeds mark the zoomed-in primary cilium. Scale bar, 10 μm, insets, 2 μm. (B and C) Quantification of ciliogenesis efficiency and length for A. Error bars, SD. n > 100 cells per experiment. Data represent mean value from three experiments per condition. Ciliation percentage: siControl = 70% ± 6, siCCDC15 = 46% ± 0.8, P = 0.0027. Cilium length: siControl = 3 μm ± 0.7, siCCDC15 = 2.3 μm ± 0.4, P < 0.0001, two-sided t test. (D) Representative U-ExM images of control and CCDC15-depleted RPE1 cells serum starved for 24 h. Centrioles and primary cilium were stained with tubulin (magenta) antibody and CCDC15 (green). Different ciliary defects associated with basal bodies efficiently depleted for CCDC15 were represented (panel on the right). Scale bar, 200 nm. (E) Representative immunofluorescence images of the effect of CCDC15 depletion on ciliary recruitment of SMO. Control and CCDC15 siRNA–depleted RPE1 cells were treated with 200 nM SAG for 24 h, fixed and stained for SMO, acetylated tubulin (Ac-tub), and DAPI. Yellow dashed boxes mark the zoomed-in primary cilium. Scale bar, 10 μm, insets, 1 μm. (F) Quantification of E. Error bars, SD. n > 50 cells per experiment. Data represent mean value from three independent experiments. SMO-positive cilia: siControl 56% ± 1, siCCDC15 25% ± 6, P = 0.0003, two-sided t test. (G) Representative images of the effect of CCDC15 depletion on basal body levels of IFT88. RPE1 cells were transfected with control or CCDC15 siRNA, fixed and stained for IFT88, acetylated tubulin, and γ-tubulin. DNA was visualized with DAPI. Yellow dashed boxes mark the zoomed-in primary cilium. Scale bar, 10 μm, insets, 1 μm. (H) Quantification of G. Graphs indicate IFT88 levels normalized to γ-tubulin levels at the basal body. Error bars, SD. n > 50 cells per experiment. Data represent the mean of three independent experiments. siControl = 1 ± 0.34, siCCDC15 = 0.67 ± 0.37. P < 0.0001, two-sided t test. ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: non-significant.

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