Cep63 Recruits Cdk1 to the Centrosome—Letter Mohammad Alsara1, Harald Lof€ ﬂer1, Anne Fechter1, Jiri Bartek2,3, and Alwin Kramer€ 1

Published OnlineFirst February 2, 2015; DOI: 10.1158/0008-5472.CAN-14-3431 Cancer Letter to the Editor Research

In 2011, we published a characterization of the phenotypes due to the size difference between Cdk1 and Cep152, the of Cep63 downregulation and overexpression in human cells in reduced Cdk1 immunoﬂuorescence signal in response to Cep63 Cancer Research (1). Recently, it was shown that several anti- depletion (Fig. 4A and B of ref. 1) might be a result of cross- bodies commonly used to detect Cdk1 cross-react with Cep152 reactivity with Cep152. (2). In our article, we used one of these antibodies (clone 17, sc- In light of this new information, it appeared to be essential to 54; Santa Cruz) to detect Cdk1 by immunoﬂuorescence and reevaluate our published results. Hence, we have repeated Cdk1 immunoblotting, and to immunoprecipitate Cdk1 (Fig. 4 of immunostaining using two antibodies shown to not cross-react ref. 1). Although our GST pulldown and, in part, our coimmu- with Cep152 (2)—ab8040 (Abcam) and 610038 (BD Bio- noprecipitation results are unaffected by this cross-reactivity sciences)—in addition to the antibody (sc-54) previously used

Figure 1. Reevaluation of centrosomal Cdk1 in Cep63- depleted and control cells. Significances were determined using the heteroscedastic, two- tailed Student t test. A, U2OS cells were transfected with siRNA to luciferase (Luc) or Cep63, respectively, fixed after 72 hours, and immunostained for g-tubulin and Cdk1 as described earlier (1). For Cdk1 immunostaining, the antibodies indicated in parentheses were used. Following the identification of centrosomes via g-tubulin signals, centrosomal Cdk1 signals were classified as strong, weak, or absent in 3 Â 100 cells per data bar. Ã,the indicated differences regarding strong centrosomal Cdk1 signals are significant (P < 0.05). B, examples of U2OS cells processed as in A and immunostained with the antibodies indicated in the figure. DNA was counterstained with DAPI (blue). Scale bars, 10 mm. C, U2OS cells were treated, immunostained, and analyzed for centrosomal Cdk1 as described in A, except that only interphase cells with separated centrosomes were included in the analysis. In 3 Â 50 cells per data bar, centrosomal Cdk1 signals were classified as strong, weak, or absent. Ã, the indicated differences regarding strong centrosomal Cdk1 signals are significant (P < 0.05). D, U2OS cells were treated, immunostained, and selected for analysis as described in C, with the ab8040 (Abcam) antibody used for Cdk1 detection. Using a Zeiss LSM 780 laser scanning microscope (Zeiss Application Center, Heidelberg), three-dimensional confocal datasets from 50 centrosomes per data bar were collected under standardized settings, and the indicated centrosomal proteins were quantified as described earlier (1). ÃÃ, the difference regarding pixel volume of centrosomal Cdk1 signals is significant (P < 0.01).

1Clinical Cooperation Unit Molecular Hematology/Oncology, Hematology/Oncology, German Cancer Research Center (DKFZ) and Depart- German Cancer Research Center (DKFZ) and Department of Med- ment of Medicine V, University of Heidelberg, Im Neuenheimer Feld 280, 2 icine V, University of Heidelberg, Heidelberg, Germany. Laboratory 69120 Heidelberg, Germany. Phone: 49-6221-42-1440; Fax: 49-6221-42-1444; of Genome Integrity and Institute of Molecular and Translational E-mail: [email protected] Medicine, Palacky University, Olomouc, Czech Republic. 3Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish doi: 10.1158/0008-5472.CAN-14-3431 Cancer Society, Copenhagen, Denmark. Corresponding Author: Alwin Kramer,€ Clinical Cooperation Unit Molecular 2015 American Association for Cancer Research.

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Alsara et al.

by us. As shown in Fig. 1A, sc-54 revealed strong centrosomal significant decrease of centrosomal Cdk1 in response to Cep63 signals in all cells treated with control siRNA, irrespective of depletion (Fig. 1D). their cell-cycle position, whereas the specific Cdk1 antibodies In conclusion, although the relative functional contribution of stained centrosomes only in a minority of control cells, which is the interaction of Cep63 with Cdk1, versus its interaction with in accordance with Cdk1 being present at centrosomes solely in Cep152, has to be carefully considered in the context of data late G2 and mitosis (3), while the uniform centrosomal immu- published by others (4, 5), our data regarding the phenotype(s) of nostaining was observed using sc-54 results from cross-reaction Cep63 downregulation and overexpression, its pathophysiologic with Cep152 (2). As reported earlier (1), centrosomal Cdk1 role, and its interaction with Cdk1 remain valid. This study, as well signals were strongly diminished in cells treated with Cep63 as the fact that the sc-54 antibody has also been used in more than siRNA when sc-54 was used for Cdk1 labeling (Fig. 1A). A 200 other publications to detect Cdk1 (2), implies that insuffi- small decrease of strong centrosomal Cdk1 signals in response cient validation even affects seemingly well-established antibo- to Cep63 depletion was also found when Cdk1-specificanti- dies and reagents and underscores the necessity to confirm data bodies were employed (Fig. 1A). Accordingly, restriction of with independent reagents as well as to post hoc amend results analysis to cells with separated centrosomes representing whenever new information questioning the validity of reagents cells in late G2 phase revealed a significant decrease of the becomes available. centrosomal Cdk1 load in response to Cep63 knockdown (Fig. 1B) with both Cdk1-specific (2) antibodies (Fig. 1C). Disclosure of Potential Conflicts of Interest fl Quantification of centrosomal Cdk1, as described in our No potential con icts of interest were disclosed. previous publication (1), using the specific ab8040 antibody Received November 24, 2014; accepted November 24, 2014; published for Cdk1 detection and restricted to late G2 cells confirmed a OnlineFirst February 2, 2015.

References € 1. Lofﬂer H, Fechter A, Matuszewska M, Saffrich R, Mistrik M, Marhold J, et al. G2/M and enters vesicles at anaphase. Embo J 1989;8:3985– Cep63 recruits Cdk1 to the centrosome: implications for regulation of 95. mitotic entry, centrosome ampliﬁcation, and genome maintenance. Cancer 4. Brown NJ, Marjanovic M, Luders€ J, Stracker TH, Costanzo V. Cep63 and Res 2011;71:2129–39. cep152 cooperate to ensure centriole duplication. PLoS ONE. 2013;8: 2. LukinaviciusG, Lavogina D, Gonczy€ P, JohnssonK. Commercial Cdk1 antibodies e69986. recognize the centrosomal protein Cep152. BioTechniques 2013;55:111–4. 5. Sir JH, Barr AR, Nicholas AK, Carvalho OP, Khurshid M, Sossick A, et al. A 3. Bailly E, Doree M, Nurse P, Bornens M. p34cdc2 is located in primary microcephaly protein complex forms a ring around parental cen- both nucleus and cytoplasm; part is centrosomally associated at trioles. Nat Genet 2011;43:1147–53.

OF2 Cancer Res; 75(4) February 15, 2015 Cancer Research

Cep63 Recruits Cdk1 to the Centrosome−−Letter

Mohammad Alsara, Harald Löffler, Anne Fechter, et al.

Cancer Res Published OnlineFirst February 2, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-14-3431

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