Eclipse t2000 e

Time-lapse microscopy was performed under a Nikon Eclipse T2000-E equipped with a mobile stage and an environmental chamber at 37°C and 5% CO₂. For the experiments involving reporter assays, plasmids were generated by fusing the Ypet ORF to the different Ccnb1 3′ UTRs. For the rescue experiments, the mouse Ccnb1 ORF was fused to either the PAS1 short or the PAS3 long 3′UTR. Linearized cDNAs were in vitro transcribed using the mMESSAGE mMACHINE T7 Transcription Kit (AM1344, Invitrogen). The in vitro transcribed mRNA coding for the reporters was microinjected along with polyadenylated mRNA coding for mCherry. The ratios of Ypet fluorescence and the maximum level of mCherry signal were used to plot the translation accumulation during time-lapse. Translation rates were calculated using Ypet/mCherry ratios as the slope obtained from simple linear regression curves from the linear portion of the time course. In the rescue experiments, oocytes were microinjected with Ccnb1 ORF fused to either PAS1 short or PAS3 long 3′UTR, and then recorded for in vitro maturation under time-lapse microscopy. Oocytes acting as controls were injected with the vehicle only.

Live cell imaging experiments were performed using a Nikon Eclipse T2000-E equipped with mobile stage and environmental chamber at 37°C and 5% CO₂. Filter set: dichroic mirror YFP/CFP/mCherry 69008BS; YFP channel (Ex: S500/20 × 49057; Em: D535/30 m 47281), mCherry channel (Ex: 580/25 × 49829; Em: 632/60 m). Images were processed and fluorescence was quantified using MetaMorph (Molecular Devices).

Collected GV-arrested oocytes were injected with 5–10 pl of 12.5 ng/μl solution of the Ypet reporters along with a mCherry reporter or FRET reporter using a FemtoJet Express programmable microinjector with an Automated Leica microinjection Microscope System (LEICA DMI 4000 B). After overnight pre-incubation in α-MEM with 1 μM cilostamide, oocytes were released from cilostamide and matured in vitro under time-lapse microscopy. Live cell imaging was performed under a Nikon Eclipse T2000-E equipped with a mobile stage and an environmental chamber at 37°C and 5% CO₂. Filter set: dichroic mirror YFP/ CFP/ mCherry 69008BS; Ypet channel (Ex: s500/20× 49057 Em: D535/30m 4728811); mCherry channel (Ex: s580/25× 49829 Em: D632/60m); Cerulean channel (Ex: 430/25× 49829 Em: 480/40m 49287), for FRET channel (Ex: 430/25× 49829 Em: D535/30m 47281). Images were processed and fluorescence was quantified using MetaMorph (Molecular Devices). For reporter assay, YFP and mCherry channels were corrected by background. Then, the ratio of Ypet fluorescence and the maximum level of mCherry signal was plotted as an accumulation of translation activity. For the FRET assay, YFP, CFP and FRET channels were corrected by background. FRET channel was corrected for the YFP bleed-trough, and FRET was calculated as FRET channel/CFP channel.