Figure 4.
CCDC15 localizes to the inner scaffold. (A) Representative confocal images of RPE1 mature centrioles expanded using U-ExM and stained for tubulin (magenta) and CCDC15 (green). Scale bar, 1 μm. (B) Top-view confocal images of RPE1 mature centriole images in U-ExM stained for tubulin in magenta and CCDC15 in green. Scale bar, 1 μm. (C) Respective lengths of tubulin and CCDC15 based on A. Error bars, SD. n > 15 centrioles from two independent experiments. Tubulin: 446 nm ± 45, CCDC15: 250 ± 41 nm. (D) Position of CCDC15 along the centriole with its respective percentage of centriole coverage, which was calculated as 55% based on C. (E) Plot profile of CCDC15 (green) and tubulin (magenta). The distance between the tubulin and CCDC15 rings were calculated as 17 nm ± 2 based on C. (F) Timing of CCDC15 centriolar recruitment during centriole duplication. Representative confocal images of RPE1 centrioles at the different stages of centriole duplication were shown. RPE1 cells synchronized in S/G2 phase were expanded using U-ExM and stained for tubulin (magenta) and CCDC15 (green). Procentriole lengths were indicated below the micrographs. Arrows mark the procentrioles. Scale bar, 1 μm. (G) Quantification of CCDC15 fluorescence intensity at different stages of centriole duplication. Normalized CCDC15 fluorescence intensity at the procentrioles was plotted against procentriole length. CCDC15 fluorescence intensity for each centriole was normalized to the mean CCDC15 fluorescence intensity of all centrioles (=1) quantified per experiment. Error bars, SD. n = 30 centrioles. Data represent mean value from three independent experiment. 0–200 nm: 0.31 ± 27, 200–400 nm: 1.23 ± 0.75, >400 nm: 1.49 ± 0.74. P < 0.0001, ns: non-signficant. P < 0.0001, two-sided t test. **** P < 0.0001, ns: non-significant. (H) Representative confocal images of CCDC15 localization (green) at the basal bodies (magenta) in RPE1 cells serum starved for 24 h and expanded using U-ExM. Scale bar, 1 μm. (I) Expression profile of CCDC15 in synchronized cells. Lysates were run on western blot and immunoblotted with CCDC15, CyclinA2, and GAPDH antibodies. U2OS cells were synchronized at the G1/S transition using a double thymidine (DT) block, then released into the cell cycle. Lysates prepared from cells at different time points were immunoblotted for CCDC15, CyclinA2 (marker for the G2/M phase), and GAPDH (loading control). Arrow marks the band for CCDC15. (J) Quantification of band intensities of CCDC15 and CyclinA2 normalized to the actin (loading control). Data represents mean value from three independent experiments. Error bars, SD. P = 0.0286, two-sided t test. *P < 0.05. Source data are available for this figure: SourceData F4.

CCDC15 localizes to the inner scaffold. (A) Representative confocal images of RPE1 mature centrioles expanded using U-ExM and stained for tubulin (magenta) and CCDC15 (green). Scale bar, 1 μm. (B) Top-view confocal images of RPE1 mature centriole images in U-ExM stained for tubulin in magenta and CCDC15 in green. Scale bar, 1 μm. (C) Respective lengths of tubulin and CCDC15 based on A. Error bars, SD. n > 15 centrioles from two independent experiments. Tubulin: 446 nm ± 45, CCDC15: 250 ± 41 nm. (D) Position of CCDC15 along the centriole with its respective percentage of centriole coverage, which was calculated as 55% based on C. (E) Plot profile of CCDC15 (green) and tubulin (magenta). The distance between the tubulin and CCDC15 rings were calculated as 17 nm ± 2 based on C. (F) Timing of CCDC15 centriolar recruitment during centriole duplication. Representative confocal images of RPE1 centrioles at the different stages of centriole duplication were shown. RPE1 cells synchronized in S/G2 phase were expanded using U-ExM and stained for tubulin (magenta) and CCDC15 (green). Procentriole lengths were indicated below the micrographs. Arrows mark the procentrioles. Scale bar, 1 μm. (G) Quantification of CCDC15 fluorescence intensity at different stages of centriole duplication. Normalized CCDC15 fluorescence intensity at the procentrioles was plotted against procentriole length. CCDC15 fluorescence intensity for each centriole was normalized to the mean CCDC15 fluorescence intensity of all centrioles (=1) quantified per experiment. Error bars, SD. n = 30 centrioles. Data represent mean value from three independent experiment. 0–200 nm: 0.31 ± 27, 200–400 nm: 1.23 ± 0.75, >400 nm: 1.49 ± 0.74. P < 0.0001, ns: non-signficant. P < 0.0001, two-sided t test. **** P < 0.0001, ns: non-significant. (H) Representative confocal images of CCDC15 localization (green) at the basal bodies (magenta) in RPE1 cells serum starved for 24 h and expanded using U-ExM. Scale bar, 1 μm. (I) Expression profile of CCDC15 in synchronized cells. Lysates were run on western blot and immunoblotted with CCDC15, CyclinA2, and GAPDH antibodies. U2OS cells were synchronized at the G1/S transition using a double thymidine (DT) block, then released into the cell cycle. Lysates prepared from cells at different time points were immunoblotted for CCDC15, CyclinA2 (marker for the G2/M phase), and GAPDH (loading control). Arrow marks the band for CCDC15. (J) Quantification of band intensities of CCDC15 and CyclinA2 normalized to the actin (loading control). Data represents mean value from three independent experiments. Error bars, SD. P = 0.0286, two-sided t test. *P < 0.05. Source data are available for this figure: SourceData F4.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal