Fig. 2.

Live cell analysis of Axl2 mRNA expression throughout the cell cycle. a Schematic representation of the tagged AXL2 gene used in live cell assays. The 24 MS2-coat-protein binding sites inserted in AXL2 5′ UTR are indicted as hairpins. The empty box and arrow represent the AXL2 gene and its transcription start site, respectively. b Impact of the MS2 tag on the abundance of Axl2 mRNA. The relative abundance of tagged and untagged mRNA was determined by qRT-PCR. The bar graphs represent the average of 3 independent biological replicates and the value of each replicate is shown as dot. c Live cell imaging of Axl2 mRNA. The MS2-tagged Axl2 mRNA was visualized, through the binding of MCP-GFP to MS2, and the images taken every 10 min are shown (for complete image sequence see Supplementary Fig. 5). The nucleus is visualized in red by the expression of td-tomato-NLS. The cell monitored in this study is indicated by arrow. The white bar equals 2 µm. The position of the RNA detected in different frames is shown at the bottom. The specificity of the spots was examined in Supplementary Fig. 4b. d Quantification of Axl2 mRNA detected in live cells. The number of RNA per cell detected every 10 min was calculated as described in the “Methods” section and plotted relative to time in minutes. The different phases of the cell cycle as function of the bud size are indicated on top. e Detecting transcriptional pulses of AXL2 in the G1 and S phases of the cell cycle. The RNA was captured every minute and plotted relative to the budding time. Budding start time (0) is indicated by vertical line. Each colored line represents data from a total of 4 cells. Trx+ and Trx− indicated transcription and no transcription detected, respectively