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. 2015 Aug 22;4:e07519. doi: 10.7554/eLife.07519

Figure 4. Centriolar satellite component CEP131 interacts with and localizes CEP152 to the centrosome to promote centriole duplication.

(A) HeLa total cell lysates were subjected to immunoprecipitation of CEP131, CEP152 and a negative control. Precipitating proteins were subjected to immunoblotting for CEP131 and CEP152. (B) HeLa cells in S phase transfected with SC or CEP131 #1 siRNA were co-stained for CEP152 (red) and Centrin (‘c’, green). (C) Total cell lysates of SC and CEP131 #1 siRNA transfected HeLa cells were analyzed by immunoblotting with antibodies to CEP131 and CEP152. Actin served as a loading control. (D) HeLa cells in S phase transfected with SC, CEP131 #1, or CEP131 #2 siRNA were co-stained for CEP131 (red) and Centrin (‘c’, green) to visualize centrioles. (E) Total cell lysates of SC, CEP131 #1, CEP131 #2 siRNA transfected HeLa cells were analyzed by immunoblotting for Cep131. α-tubulin served as a loading control. 20 μg of protein lysate was loaded per lane. (F) Quantification of the mean percentage of SC, CEP131 #1, or CEP131 #2 siRNA transfected cells in S phase with four centrioles. (G) SC and CEP131-depleted S phase cells were co-stained with Centrin (‘c’, green), CDK5RAP2 (red), CEP152 (red), WDR62 (red), and CEP63 (red). (H) Schematic indicating that CEP131 is required to localize CEP152, WDR62 and CEP63 to the centrosome. For all quantifications at least 100 cells were counted per experiment (n = 3), p < 0.005 (paired t-test). Scale bars indicate 5 μm for all images.

DOI: http://dx.doi.org/10.7554/eLife.07519.017

Figure 4.

Figure 4—figure supplement 1. Cep131gt/gt MEFs exhibit centriole duplication and centrosome organizational defects.

Figure 4—figure supplement 1.

(A) WT and two Cep131gt/gt MEF cell lines were analyzed by immunofluorescence with antibodies to Centrin (‘c’, green) and γ-tubulin (red). (B) Quantification of the number of centrioles in asynchronous populations of WT and two Cep131gt/gt MEFs. (C) Immunofluorescence of WT and Cep131gt/gt MEFs co-stained with Centrin (‘c’, green) and CEP152 (red). For all quantifications at least 100 cells were counted per experiment (n = 3). p < 0.005 (paired t-test) compared to WT was denoted with an asterisk. Scale bars indicate 5 μm for all images.
DOI: http://dx.doi.org/10.7554/eLife.07519.018
Figure 4—figure supplement 2. CEP131 is required for the centrosomal localization of CEP152, WDR62 and CEP63.

Figure 4—figure supplement 2.

(A) Quantification of the mean fluorescence intensities ±s.d. of CDK5RAP2, CEP152, WDR62 and CEP63 in SC or CEP131-depleted cells expressed as the mean percentage ±s.d. of the fluorescence intensities of SC cells. For all quantifications 30 cells were analyzed per experiment (n = 3). p < 0.005 (paired t-test) statistically significant differences are denoted by an asterisk. (B) Total cell lysate from SC and CEP131 siRNA-treated HeLa cells were analyzed by immunoblotting using antibodies to CEP131, CDK5RAP2, CEP152, WDR62, and CEP63. Actin served as a loading control.
DOI: http://dx.doi.org/10.7554/eLife.07519.019
Figure 4—figure supplement 3. CEP152 associates with CDK5RAP2 and WDR62 in a CEP131-dependent manner.

Figure 4—figure supplement 3.

(A) Total cell lysate from SC and CEP131-depleted HeLa cells were subjected to immunoprecipitation using an antibody to CEP152. Co-precipitating proteins were analyzed by immunoblotting for CDK5RAP2 and WDR62. SC and CEP131 siRNA transfected HeLa cell total cell lysates were analyzed by immunoblotting with antibodies to CDK5RAP2, CEP152, WDR62 and CEP131. Actin served as a loading control.
DOI: http://dx.doi.org/10.7554/eLife.07519.020