Cell Cycle Dynamics Visualization

To monitor the cell-cycle, cell division and cell movement, GFP-labeled primary mesothelial cells from CR−/− and WT mice were infected with a lentivirus coding for mCherry-hCdt1 (30/120) [18 (link), 19 (link)]. The Fucci (Fluorescent, ubiquitination-based cell cycle indicator) mCherry-hCdt1 was a kind gift of Prof. H. Miyoshi (Riken, Japan). mCherry-hCdt1 was synthetized by PCR with the forward primer (FW-PmeI-mCherry 5’-AGT CGT TTA AAC ATG GTG AGC AAG GGC GAG GAG-3’) and reverse primer (RV-SpeI-mCherry 5’-AGT CAC TAG TTT AGA TGG TGT CCT GGT CCT G-3’) and cloned into pLVTHM (Addgene plasmid #12247) (SpeI and PmeI sites) substituting eGFP. pLV-mCherry-hCdt1 was used to produce lentivirus. The expression level of mCherry-hCdt1 is cell cycle-dependent showing an accumulation during the G₀/G₁ phase, followed by an ubiquitination–based protein degradation during S/G₂/M phases. Fluorescence and time-lapse microscopy was performed with an inverted fluorescence microscope DMI 6000B (Leica Microsystems) equipped with an incubation chamber. A digital camera (Leica), a 10× objective, GFP and TXR filter sets and the LAS-AF imaging software (Leica) were used to acquire the images. Images were taken every hour using the same settings including exposure times, gains and positions. For the image analysis a Java ImageJ plugin: CGE [17 (link)] was used.