Generation and characterization of stable cell lines expressing WT or mutant GFP-CPAP. (A) Sequencing results of the genomic mutation using gel-purified PCR products. (B) Western blots illustrating that the Flp-In–induced protein expression system has a low level of leaky expression, where CPAP endogenous (endo) is compared with CPAP overexpression (OE). (C) Western blots illustrating the CPAP expression levels in control, host, and different GFP-CPAP-FLWT and GFP-CPAP-FLMUT cells lines without doxycycline induction. (D) Immunofluorescence images taken with Airyscan 2 confocal microscope of centrioles of cells blocked for 24 h in mitosis with S-trityl-L-cysteine (STLC) and stained for the acetylated tubulin (blue), CP110 (green), and GFP-CPAP (magenta). (E) Median ± IQR of centriole length in mitotically blocked cells by STLC, measured as in Fig. 8 F. Number of analyzed mother centrioles (MC) and daughter centrioles (DC): control cells, n = 74 MC, 53 DC; host, n = 71 MC, 44 DC; CPAP-FLWT#3, n = 66 MC, 69 DC, and CPAP-FLMUT#1, n = 50 MC, 40 DC; and nonsignificant (ns) calculated using Kruskal–Wallis ANOVA test. (F and G) U-ExM images of centrioles from host, CPAP-FLWT#4, and CPAP-FLMUT#5 cells blocked in G1/S and stained for acetylated tubulin (blue) combined with CP110 (green) in F and CPAP (magenta) in G. (F) Normal centrioles from host and CPAP-FLWT#4 and incomplete centriole from CPAP-FLMUT#5. (G) Normal centrioles from host and CPAP-FLWT#4 and incomplete centriole from CPAP-FLMUT#5. Scale bar is corrected for ∼4.5 expansion factor. Source data are available for this figure: SourceData FS5.
