Sei1 in Cancer and Diabetes Figure 2. Cell cycle of Sei1-null cells. (A) Cell extracts from asynchronously growing MEFs of the indicated genotypes were immunoprecipitated using cyclinD1 or cyclinD2 antibodies, and then immunoblotted against Cdk4 to assess Cdk4-cyclinD complex formation. (B, C and D) MEF cultures of the indicated genotypes were synchronized by serum withdrawal, and forced to re-enter the cell cycle by addition of serum to the medium. (B) Percentage of cells in S phase was determined by the incorporation of propidium iodide and FACS analysis. The graph represents the average and s.d. of at least two independent preparations of primary MEFs per genotype. ANOVA analysis (using Sidak multiple testing) indicated that Cdk4-null MEFs were significantly different (p,0.05) than wt MEFs at all time points. Sei1-null MEFs only showed a significant difference with wt MEFs at time 8 h. (C) Representative example of two experiments measuring Rb phosphorylation by immunoblotting protein extracts obtained at the indicated times. (D) Representative example of at least two experiments assaying Cdk2 kinase activity from protein extracts of synchronyzed MEFs at the indicated times; asynch.: MEFs asynchronously growing. (E) Splenocytes isolated from at least three mice per genotype were activated by LPS and concanavalin A, and the number of lymphocytes was counted at the indicated times. Values correspond to the average and s.d. doi:10.1371/journal.pone.0008744.g002 phosphorylation of Rb during the first cell cycle after serum re- addition, and observed no significant differences between wt and Sei1-null cells (Fig. 2C and Supp. Fig. S3B). Finally, we also assessed the activation of Cdk2 and, again, the kinetics and intensity of activation of this kinase were similar across various assays when comparing wt and Sei1-null MEFs (Fig. 2D and Supp. Fig. S3C). To widen the scope of the previous analyses, we checked cell cycle entry in splenocytes, which include both T- and B-lymphocytes. These cells remain quiescent in culture until activated with antigens. Sei1-null splenocytes entered proliferation with kinetics and efficiencies indistinguishable from wt splenocytes (Fig. 2E). Sei1 has been also described as a positive co-factor for the transcriptional regulator E2f1 [15]. We examined E2f1-driven transcription using a luciferase reporter assay (based on the E2f1 promoter, [20]). However, the absence of Sei1 did not affect the expression of luciferase by endogenous E2f1 levels (Fig. 3A) or by overexpressed transfected E2f1 (Fig. 3B). Finally, we checked the activity of E2f1 by measuring the expression of a well known E2f1 target, namely p19Arf [21]. Retroviral expression of E2f1 efficiently upregulated p19Arf, but no differences were observed between wt and Sei1-null cells (Fig. 3C). Also, under basal conditions the levels of p19Arf were similar regardless of the Sei1 genotype (Fig. 3C). All together, we conclude that the absence of Sei1 does not affect significantly the cell cycle, neither in MEFs nor in lymphocytes, and does not impair detectably the assembly of Cdk4 complexes or the transcriptional activity of E2f1 in fibroblasts. Normal Immortalization and Oncogenic Transformation of Sei1-Null Cells Cell cycle regulators may have profound effects on the susceptibility of cells to acquire oncogenic properties [1]. Thus, we studied the serum dependence, immortalization and transfor- mation capacity of MEFs deficient for Sei1. As shown in Fig. 4A, Sei1-null MEFs proliferate at the same rate as wt MEFs under different serum concentrations. When subjected to the 3T3 serial passage protocol, we could not observe differences in the onset of senescence or in the immortalization of wt and Sei1-null MEFs (Fig. 4B). To further test this under more stringent conditions, we seeded wt and Sei1-null cells at low cellular density and checked for the appearance of colonies. Sei1-null MEFs showed the same low colony formation capacity as wt MEFs, while Ink4a/Arf- double MEFs, used here as a positive control, formed colonies efficiently (Fig. 4C). Finally, we examined the impact of Sei1 deficiency on the susceptibility of MEFs to neoplastic transforma- PLoS ONE | www.plosone.org 3 January 2010 | Volume 5 | Issue 1 | e8744