Fig. 2. Phenotypic characterization of Env-induced karyogamy. (A) Cell cycle-relevant proteins whose expression level changes (by a factor of ≥2) upon syncytium formation as determined by quantitative immunoblots (X ± SEM, n = 3). (B) Examples of cell cycle-relevant proteins detectable in HeLa cells whose expression level varies by a factor <2. (C) 5-bromo-2′deoxyuridine (BrdU) incorporation into syncytia as a function of the karyogamy (KG) status. Sixteen-hour-old syncytia were incubated with BrdU for 2 h and then subjected to the immunodetection of BrdU into S-phase nuclei. (D) Tubulin staining of 18-h-old syncytia. Note that the tubulin network is excluded from the nucleus before karyogamy. After karyogamy, nuclei stain positively for tubulin. (E) Lamin B immunostaining of syncytia. Note that karyogamic syncytia exhibit the breakdown of lamin B. (F) Loss of nuclear envelope barrier function in karyogamy. Syncytia were microinjected into the cytoplasm with dextran-70 FITC conjugated, which is excluded from the nucleus of pre-karyogamic cells, yet penetrates into the nucleus of karyogamic syncytia. (G) Cyclin B accumulation in pre-karyogamic syncytia. Two phenotypes (12 h after fusion) are shown, namely pre-karyogamic (p53^S15P⁺) cells exhibiting cyclin B in the cytoplasm or in the nucleus. In contrast, most karyogamic cells lack immunodetectable cyclin B. The kinetics of cyclin B accumulation is shown at different intervals. The few karyogamic (p53^S15P⁺) cells positively staining for cyclin B demonstrate a diffuse (cytoplasmic + nuclear) staining pattern. (H) Kinetics of cyclin B accumulation and the phosphorylation of p53^S15 or Cdk1T15. (I) Kinetics of Cdk1 activity. Cdk1 was immunoprecipitated at different intervals after syncytium formation and its capacity to phosphorylate histone H1 in vitro was determined (H1-P*) in the absence or presence of roscovitine (1 µM). Immunoblot detection to confirm equal loading of Cdk1 was also performed.