Figure S4.
CCDC15 is required for centriole amplification, but not canonical centriole duplication. (A) Immunofluorescence validation of CCDC15 depletion by siRNA treatment in RPE1 cells. RPE1 cells were transfected with control and CCDC15 siRNAs. 96 h after transfection, cells were fixed with methanol and stained for CCDC15 and γ-tubulin. Error bars, SD. n > 100 cells per experiment. Data represent mean value from three experiments per condition. siControl: 1 ± 0.43, siCCDC15: 0.56 ± 0.55, P < 0.0001, two-sided t test. Scale bar, 10 μm, insets, 2 μm. Statistical analysis was done by normalizing the values to the mean of siControl. Absolute intensity for CCDC15 was not plotted. Instead, intensities were normalized to the average CCDC15 intensity of the control sample. (B) Validation of siRNA-mediated depletion of POC5, POC1B, and FAM161A in RPE1 cells. Cells were transfected with control or POC5, POC1B, and FAM161A siRNAs and extracts from these cells were immunoblotted for the indicated proteins and vinculin as loading control. (C) Cell cycle profile of control and CCD15-depleted RPE1 cells. RPE1 cells were transfected with control and CCDC15 siRNAs. 96 h after transfection, cells were fixed with ethanol and stained with Muse Cell Cycle kit. Error bars, SD. Data represent mean value from two independent experiments per condition. For G0/G1 phase, siControl = 74.20% ± 2.12, siCCDC15 = 79.20% ± 2.83, P = 0.1835%; for S phase, siControl = 6.95% ± 0.78, siCCDC15 = 7.8% ± 0.42, P = 0.3077; for G2/M phase, siControl = 17.80% ± 1.70, siCCDC15 = 13.60% ± 2.12, P = 0.1603, one-way ANOVA. (D) Quantification of centriole number in control or CCDC15 siRNA–transfected asynchronous RPE1 cells. Error bars, SD. n > 100 cells per experiment. Data represent mean value from two independent experiments per condition. Centriole number >4 siControl = 0.71% ± 0.3, siCCDC15 = 0.62% ± 0.5, P = 0.7397, two-sided t test. (E) Representative immunofluorescence images of control and CCDC15-depleted cells stained for SAS6, PCNA, and Centrin-2. DNA was visualized with DAPI. Scale bar, 10 μm, insets, 2 μm. (F) Quantification of SAS6 dots in PCNA-positive cells in D. Error bars, SD. n > 50 cells per experiment. Data represent mean value from three experiments per condition. SAS6 2 dots: siControl = 93% ± 3, siCCDC15 = 88% ± 4, P = 0.1565; SAS6 1 dot: siControl = 8% ± 1, siCCDC15 = 12% ± 4, P = 0.2172, two-sided t test. (G) Representative images of centrioles in control and CCDC15-depleted RPE1 cells synchronized by STLC treatment. Cells were transfected with control and CCDC15 siRNA and treated with 50 µM STLC for 18 h before fixation. Cells were then stained for CCDC15 and Centrin-2. The DNA was visualized with DAPI. Scale bar, 10 μm. (H) Quantification of cells with more than four centrioles based on F. Error bars, SD. n > 100 cells per experiment. Data represent mean value from three experiments per condition. siControl = 95% ± 1, siCCDC15 = 63% ± 4, P = 0.0002, two-sided t test. (I) CCDC15 depletion compromises S phase arrest overduplication of centrioles. U2OS cells were transfected with control siRNA or CCDC15 siRNA and arrested in S phase by hydroxyurea treatment for 48 h. Cells were then stained with CCDC15 and Centrin-3. DNA was visualized with DAPI. Scale bar, 10 μm, insets, 2 μm. (J) Quantification of cells with >4 centrioles based on H. Error bars, SD. n > 100 cells per experiment. Data represent mean value from two experiments per condition. siControl = 42% ± 1, siCCDC15 = 28% ± 2, P = 0.0148, two-sided t test. (K) CCDC15 depletion compromises PLK4-induced centriole amplification. RPE-1 cells stably expressing Tet-inducible Plk4 were depleted of CCDC15 by siRNA for 72 h then treated with doxycycline for 18 h to induce Plk4 expression. Cells were fixed and stained for PLK4, Centrin-2, and γ-tubulin. DNA was visualized with DAPI. Scale bar, 10 μm. (L) Quantification of cells with more than four centriole dots based on J. Error bars, SD. n > 100 cells per experiment. Data represent mean value from three experiments per condition. siControl = 83% ± 9, siCCDC15 = 41% ± 6, P = 0.0024, two-sided t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: non-significant. Source data are available for this figure: SourceData FS4.

CCDC15 is required for centriole amplification, but not canonical centriole duplication. (A) Immunofluorescence validation of CCDC15 depletion by siRNA treatment in RPE1 cells. RPE1 cells were transfected with control and CCDC15 siRNAs. 96 h after transfection, cells were fixed with methanol and stained for CCDC15 and γ-tubulin. Error bars, SD. n > 100 cells per experiment. Data represent mean value from three experiments per condition. siControl: 1 ± 0.43, siCCDC15: 0.56 ± 0.55, P < 0.0001, two-sided t test. Scale bar, 10 μm, insets, 2 μm. Statistical analysis was done by normalizing the values to the mean of siControl. Absolute intensity for CCDC15 was not plotted. Instead, intensities were normalized to the average CCDC15 intensity of the control sample. (B) Validation of siRNA-mediated depletion of POC5, POC1B, and FAM161A in RPE1 cells. Cells were transfected with control or POC5, POC1B, and FAM161A siRNAs and extracts from these cells were immunoblotted for the indicated proteins and vinculin as loading control. (C) Cell cycle profile of control and CCD15-depleted RPE1 cells. RPE1 cells were transfected with control and CCDC15 siRNAs. 96 h after transfection, cells were fixed with ethanol and stained with Muse Cell Cycle kit. Error bars, SD. Data represent mean value from two independent experiments per condition. For G0/G1 phase, siControl = 74.20% ± 2.12, siCCDC15 = 79.20% ± 2.83, P = 0.1835%; for S phase, siControl = 6.95% ± 0.78, siCCDC15 = 7.8% ± 0.42, P = 0.3077; for G2/M phase, siControl = 17.80% ± 1.70, siCCDC15 = 13.60% ± 2.12, P = 0.1603, one-way ANOVA. (D) Quantification of centriole number in control or CCDC15 siRNA–transfected asynchronous RPE1 cells. Error bars, SD. n > 100 cells per experiment. Data represent mean value from two independent experiments per condition. Centriole number >4 siControl = 0.71% ± 0.3, siCCDC15 = 0.62% ± 0.5, P = 0.7397, two-sided t test. (E) Representative immunofluorescence images of control and CCDC15-depleted cells stained for SAS6, PCNA, and Centrin-2. DNA was visualized with DAPI. Scale bar, 10 μm, insets, 2 μm. (F) Quantification of SAS6 dots in PCNA-positive cells in D. Error bars, SD. n > 50 cells per experiment. Data represent mean value from three experiments per condition. SAS6 2 dots: siControl = 93% ± 3, siCCDC15 = 88% ± 4, P = 0.1565; SAS6 1 dot: siControl = 8% ± 1, siCCDC15 = 12% ± 4, P = 0.2172, two-sided t test. (G) Representative images of centrioles in control and CCDC15-depleted RPE1 cells synchronized by STLC treatment. Cells were transfected with control and CCDC15 siRNA and treated with 50 µM STLC for 18 h before fixation. Cells were then stained for CCDC15 and Centrin-2. The DNA was visualized with DAPI. Scale bar, 10 μm. (H) Quantification of cells with more than four centrioles based on F. Error bars, SD. n > 100 cells per experiment. Data represent mean value from three experiments per condition. siControl = 95% ± 1, siCCDC15 = 63% ± 4, P = 0.0002, two-sided t test. (I) CCDC15 depletion compromises S phase arrest overduplication of centrioles. U2OS cells were transfected with control siRNA or CCDC15 siRNA and arrested in S phase by hydroxyurea treatment for 48 h. Cells were then stained with CCDC15 and Centrin-3. DNA was visualized with DAPI. Scale bar, 10 μm, insets, 2 μm. (J) Quantification of cells with >4 centrioles based on H. Error bars, SD. n > 100 cells per experiment. Data represent mean value from two experiments per condition. siControl = 42% ± 1, siCCDC15 = 28% ± 2, P = 0.0148, two-sided t test. (K) CCDC15 depletion compromises PLK4-induced centriole amplification. RPE-1 cells stably expressing Tet-inducible Plk4 were depleted of CCDC15 by siRNA for 72 h then treated with doxycycline for 18 h to induce Plk4 expression. Cells were fixed and stained for PLK4, Centrin-2, and γ-tubulin. DNA was visualized with DAPI. Scale bar, 10 μm. (L) Quantification of cells with more than four centriole dots based on J. Error bars, SD. n > 100 cells per experiment. Data represent mean value from three experiments per condition. siControl = 83% ± 9, siCCDC15 = 41% ± 6, P = 0.0024, two-sided t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: non-significant. Source data are available for this figure: SourceData FS4.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal