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. 2022 Jul 5;11:e77892. doi: 10.7554/eLife.77892

Figure 2. Molecular composition of caMTOCs in AKAP450/CAMSAP2 knockout cells.

All the cells used in this figure are AKAP450/CAMSAP2 knockout cells, except for panel E as indicated. (A–C) Immunofluorescence images of control or centrinone-treated AKAP450/CAMSAP2 knockout RPE1 cells stained for and depleted of the indicated proteins. (D, F–I) Immunofluorescence images of centrinone-treated AKAP450/CAMSAP2 knockout RPE1 cells stained for and depleted of the indicated proteins. (E) Immunofluorescence images of centrinone-treated AKAP450/CAMSAP2/p53 knockout and AKAP450/CAMSAP2/p53/pericentrin knockout RPE1 cells stained as indicated. In panels A-I, insets show enlargements of the merged channels of the boxed areas, and dashed lines indicate cell edges. (J) Quantification of the main PCM organization types, as described in Figure 1—figure supplement 2A, for cells prepared as described in panel A-C, E-I. Numbers on the histogram show the percentages. 1293 (-CentB), 1547(+CentB), 2021(siLuci), 1822(siCEP152), 1345(siCEP192), 1161(siNEDD1), 2302 (AKAP450/CAMSAP2/p53/PCNT knockout (PCNT KO)), 2264(siCDK5RAP2), 2510(siγ-tubulin), 2408(siNIN) and 2526(siDHC) cells were analyzed for each measurement in three independent experiments (n=3). Values represent mean ± SD. (K) Summarizing table of PCM localization and the depletion effects on caMTOC formation in AKAP450/CAMSAP2 knockout RPE1 cells. NT, not tested.

Figure 2—source data 1. An Excel sheet with numerical data on the quantifications shown in panel J.

Figure 2.

Figure 2—figure supplement 1. Characterization of PCM components localizing to caMTOCs in AKAP450/CAMSAP2 knockout cells.

Figure 2—figure supplement 1.

(A) Western blot showing that endogenous NEDD1 and CEP192 are present in centrinone-treated AKAP450/CAMSAP2 knockout cells. (B) Western blot results showing the depletion of the indicated PCM proteins. (C) Immunofluorescence images of centrinone-treated AKAP450/CAMSAP2 knockout RPE1 cells showing the localization and the effects of depletion of the indicated proteins on the caMTOCs. siRNA against luciferase was used as a control. Zooms show enlargements of the merged channels of the boxed areas.
Figure 2—figure supplement 1—source data 1. Full raw unedited western blots shown in panels A and B.
Figure 2—figure supplement 2. Generation of the AKAP450/CAMSAP2/p53/pericentrin knockout RPE1 cell line.

Figure 2—figure supplement 2.

(A) Diagram illustrating two sgRNA sites targeting p53 exon 2 and exon 4. The green and red boxes indicate the position of the PAM sites, and the predicted Cas9 cut sites are indicated by scissors. (B,C) Genotyping results of the genomic mutation using gel-purified PCR product which covers exon 2 and exon 4 of the p53-encoding gene in AKAP450/CAMSAP2/p53 knockout cell line and a diagram illustrating the induced inversion. (D) Diagram illustrating the binding sites of p53 antibodies and the position of the mutation induced in p53-encoding gene. (E,F) Immunofluorescence and Western blot images showing p53 expression in control and AKAP450/CAMSAP2/p53 knockout cells. (G–H) Genotyping results of the genomic mutation using gel-purified PCR product which covers the two sgRNAs targeting sites within pericentrin-encoding gene in AKAP450/CAMSAP2/p53/pericentrin knockout cell line and a diagram illustrating the induced genomic mutations within exon 5 of the pericentrin-encoding gene. The green and red boxes indicate the position of the PAM sites, and the predicted Cas9 cut sites are indicated by scissors. (I) Diagram illustrating the binding domain of the pericentrin antibody and the positions of the mutations induced downstream of the Cas9 cutting sites. (J) Immunofluorescence images confirming the loss of pericentrin in AKAP450/CAMSAP2/p53/pericentrin knockout cells. (K) Western blot confirming the loss of p53 and pericentrin in AKAP450/CAMSAP2/p53/pericentrin knockout cells.
Figure 2—figure supplement 2—source data 1. Full raw unedited western blots shown in panels F and K.
Figure 2—figure supplement 3. Characterization of cell division in centrinone-treated AKAP450/CAMSAP2/p53 knockout cells.

Figure 2—figure supplement 3.

(A) Immunofluorescence images of metaphase AKAP450/CAMSAP2/p53 knockout cells treated with DMSO and centrinone (CentB) and stained for h microtubules (α-tubulin, magenta), PCM (PCNT, red), centrioles (Centrin, green) and DNA (DAPI, blue). (B–C) Phase-contrast time-lapse images of centrinone-treated AKAP450/CAMSAP2/p53 knockout RPE1 cells. Time is hh:mm. (B) The red and blue arrows show two cells with different mitosis duration. (C) Mitotic phases in a representative cell. (D) Quantification of the mitosis duration of centrinone-treated AKAP450/CAMSAP2/p53 knockout RPE1 cells. Thirty-six cells from 14 videos were analyzed. Values represent mean ± SD.
Figure 2—figure supplement 3—source data 1. An Excel sheet with numerical data on the quantifications shown in panel D.
Figure 2—figure supplement 4. Characterization of PCM organization in AKAP450/CAMSAP2/p53/pericentrin knockout cells.

Figure 2—figure supplement 4.

(A) Immunofluorescence images of centrinone-treated AKAP450/CAMSAP2/p53 knockout (Control) and AKAP450/CAMSAP2/p53/pericentrin knockout (PCNT KO) RPE1 cells showing staining for the indicated proteins. Zooms of the boxed areas show merged channels. (B) Western blots showing the expression levels of CDK5RAP2, γ-tub, and ninein in control (+DMSO) and centrinone-treated (+CentB) AKAP450/CAMSAP2/p53 knockout and AKAP450/CAMSAP2/p53/PCNT knockout RPE1 cells.
Figure 2—figure supplement 4—source data 1. Full raw unedited western blots shown in panel B.
Figure 2—figure supplement 5. Effects of the depletion or knockout of EB1 and EB3 on caMTOC formation in AKAP450/CAMSAP2 knockout cells.

Figure 2—figure supplement 5.

(A) Immunofluorescence images of centrinone-treated AKAP450/CAMSAP2 knockout cells stained for EB1 or EB3 and pericentrin showing effects of EB1 or EB3 depletion on caMTOCs. (B) Western blot confirming the loss of AKAP450 and CAMSAP2 in AKAP450/CAMSAP2/EB1/EB3 mutant cells. (C) Immunofluorescence images of control or centrinone-treated AKAP450/CASMSAP2/EB1/EB3 mutant RPE1 cells stained for the indicated proteins. Zooms of the boxed areas show merged channels.
Figure 2—figure supplement 5—source data 1. Full raw unedited western blots shown in panel B.