Fig. 4. Mos causes G1 arrest in starfish blastomeres. (A,B) GST-Mos injection activates MAPK and Rsk, and causes cell cycle arrest at interphase in blastomeres. Mature eggs 2.5 hours after 1-MeAde addition were inseminated. At the two-cell stage (around 1.5-2.0 hours after insemination), one cell was injected with 25 pg of GST-Mos or control GST, or not injected, and the embryos were further cultured for 5-5.5 hours. At indicated times, lysates from whole embryos were immunoblotted with anti-MAPK antibody, or immunoprecipitated with anti-Rsk serum 1 for phosphorylation of GST-S6 (A). Whereas the non-injected half developed to the early blastula stage, nuclei were observed in the GST-Mos-injected half of large cells, indicating the cell cycle arrest at interphase (B, right). (C) GST-Mos causes G1 arrest after fertilization. Mature eggs were inseminated, and 20-30 minutes later (around entry into the first S-phase), were injected with GST-Mos or control GST, or not injected. After an additional 30 minutes (around entry into the first M-phase in control eggs), eggs were incubated for 3 hours in the presence of BrdU to examine DNA replication. In control eggs injected with GST, although significant delay in cell cycle progression was observed, BrdU was clearly incorporated into DNA. By contrast, no incorporation of BrdU was seen in GST-Mos-injected eggs, indicating cell cycle arrest at G1 phase. The numbers of eggs examined are indicated in parentheses.