Figure 5.
CCDC15 depletion leads to shorter and structurally aberrant centrioles. (A) CCDC15 depletion leads to shorter centrioles. Representative confocal images of expanded centrioles from control and CCDC15-depleted cells stained for CCDC15 (green) and tubulin (magenta). RPE1 cells were transfected with control or CCDC15 siRNA. 96 h after transfection, cells were expanded by U-ExM. Note that CCDC15 was efficiently depleted from only one of the centrioles in most cells (white arrow). Scale bar, 200 nm. (B) Quantification of percentage of CCDC15-positive centrioles based on A. n > 40 centrioles per experiment. Data represents mean value from three independent experiments. Error bars, SD. siControl = 100%, siCCDC15 = 48% ± 4. P = 0.0031, two-sided t test. (C) Centriole length quantification based on A. Error bars, SD. n > 10 centrioles per experiment. Data represents mean value from three independent experiments. siControl: 413 nm ± 66, siCCDC15: 367 nm ± 62. P = 0.0001, two-sided t test. Centrioles depleted of CCDC15 were 45 nm shorter compared with control centrioles in RPE1 cells. (D) Changes in diameter in distal (d), central (c), and proximal (p) regions of the centrioles. RPE1 cells were transfected with control or CCDC15 siRNA and expanded by U-ExM 96 h after transfection. Gels were stained with tubulin (magenta) and endogenous CCDC15 (green) antibodies. Scale bar, 200 nm. (E) Quantification of D. Error bars, SD. n > 10 centrioles per experiment. Data represents mean value from three independent experiments. Distal region: siControl: 222 nm ± 26, siCCDC15: 232 nm ± 25, P = 0.0828; core region siControl: 233 nm ± 23, siCCDC15: 247 nm ± 28, P = 0.0204; proximal region siControl: 242 nm ± 23, siCCDC15: 242 nm ± 27, P = 0.9856, two-sided t test. (F) Representative confocal images of expanded centrioles in control and CCDC15-depleted RPE1 cells stained for CCDC15 (green) and tubulin (magenta). Different types of structural defects of CCDC15-depleted cells were represented, which included centrioles with broken, wider or shorter microtubule walls. Scale bar, 200 nm. (G) Depletion of POC1B and POC5 lead to shorter centrioles while FAM161A has no effect. Representative confocal images of expanded centrioles from control, POC1B, POC5, and FAM161A-depleted cells stained for tubulin (magenta). RPE1 cells were transfected with control, POC1B, POC5, and FAM161A siRNA. 48 h for POC1B and POC5 and 76 h for FAM161A after transfection, cells were expanded by U-ExM. Scale bar, 200 nm. (H) Centriole length quantification based on G. Error bars, SD. n > 10 centrioles per experiment. Data represents mean value from three independent experiments. POC1B: siControl: 400.6 nm ± 36.1, siPOC1B: 341.5 ± 44.39 nm, P < 0.0001; POC5: siControl: 414.1 nm ± 38.3, siPOC5: 432.7 nm ± 44.8, P = 0.0647, FAM161A: siControl: 447.8 nm ± 59.7, siFAM161A: 436.3 nm ± 64, P = 0.3549, two-sided t test. * P < 0.05, *** P < 0.001, **** P < 0.0001, ns: non-significant.

CCDC15 depletion leads to shorter and structurally aberrant centrioles. (A) CCDC15 depletion leads to shorter centrioles. Representative confocal images of expanded centrioles from control and CCDC15-depleted cells stained for CCDC15 (green) and tubulin (magenta). RPE1 cells were transfected with control or CCDC15 siRNA. 96 h after transfection, cells were expanded by U-ExM. Note that CCDC15 was efficiently depleted from only one of the centrioles in most cells (white arrow). Scale bar, 200 nm. (B) Quantification of percentage of CCDC15-positive centrioles based on A. n > 40 centrioles per experiment. Data represents mean value from three independent experiments. Error bars, SD. siControl = 100%, siCCDC15 = 48% ± 4. P = 0.0031, two-sided t test. (C) Centriole length quantification based on A. Error bars, SD. n > 10 centrioles per experiment. Data represents mean value from three independent experiments. siControl: 413 nm ± 66, siCCDC15: 367 nm ± 62. P = 0.0001, two-sided t test. Centrioles depleted of CCDC15 were 45 nm shorter compared with control centrioles in RPE1 cells. (D) Changes in diameter in distal (d), central (c), and proximal (p) regions of the centrioles. RPE1 cells were transfected with control or CCDC15 siRNA and expanded by U-ExM 96 h after transfection. Gels were stained with tubulin (magenta) and endogenous CCDC15 (green) antibodies. Scale bar, 200 nm. (E) Quantification of D. Error bars, SD. n > 10 centrioles per experiment. Data represents mean value from three independent experiments. Distal region: siControl: 222 nm ± 26, siCCDC15: 232 nm ± 25, P = 0.0828; core region siControl: 233 nm ± 23, siCCDC15: 247 nm ± 28, P = 0.0204; proximal region siControl: 242 nm ± 23, siCCDC15: 242 nm ± 27, P = 0.9856, two-sided t test. (F) Representative confocal images of expanded centrioles in control and CCDC15-depleted RPE1 cells stained for CCDC15 (green) and tubulin (magenta). Different types of structural defects of CCDC15-depleted cells were represented, which included centrioles with broken, wider or shorter microtubule walls. Scale bar, 200 nm. (G) Depletion of POC1B and POC5 lead to shorter centrioles while FAM161A has no effect. Representative confocal images of expanded centrioles from control, POC1B, POC5, and FAM161A-depleted cells stained for tubulin (magenta). RPE1 cells were transfected with control, POC1B, POC5, and FAM161A siRNA. 48 h for POC1B and POC5 and 76 h for FAM161A after transfection, cells were expanded by U-ExM. Scale bar, 200 nm. (H) Centriole length quantification based on G. Error bars, SD. n > 10 centrioles per experiment. Data represents mean value from three independent experiments. POC1B: siControl: 400.6 nm ± 36.1, siPOC1B: 341.5 ± 44.39 nm, P < 0.0001; POC5: siControl: 414.1 nm ± 38.3, siPOC5: 432.7 nm ± 44.8, P = 0.0647, FAM161A: siControl: 447.8 nm ± 59.7, siFAM161A: 436.3 nm ± 64, P = 0.3549, two-sided t test. * P < 0.05, *** P < 0.001, **** P < 0.0001, ns: non-significant.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal