Fig. 2. Characterization of OSCAR in vitro.

a Design of the OSCAR ratiometric reporter vector. Wild-type mCherry and VeN90 are expressed from a single mRNA encoding a self-cleaving P2A peptide and a nuclear localization signal fused to VeN90. During translation, mCherry and VeN90 proteins are cleaved in a 1:1 ratio allowing for normalization of VeN90 expression even if VeN90 is phosphorylated by CDK9/CCNT1 and dim in active cells. Absence of CDK9/CCNT1 in dormant cells results in gain of green fluorescence due to VeN90 lack of phosphorylation. Transcriptionally low (dormant) cells thus appear more green that non-dormant cells. Low transcription in dormant cells leads to reduced mRNA expression of the reporter leading to low mCherry and VeN90 protein expression, but reduced phosphorylation of VeN90 enhances (or rescues) its fluorescence intensity. b Representative image of a mouse small intestinal organoid infected with a lentivirus encoding OSCAR. After infection, the organoid was fixed, permeabilized and stained for RNApII-pSer2 and DAPI and visualized for endogenous mCherry and VeN90 fluorescence. Scale bar = 20 μM. c Enlarged image corresponding to white box in (b). Empty arrowheads indicate cells with high VeN90/Low RNApII-pSer2, while filled arrowheads indicate RNApII-pSer2-positive cells showing low VeN90 fluorescence. d Single cell quantification of the fluorescence signal of all fluorescent cells in (b) shows inverse correlation of OSCAR (VeN90/mCherry) to RNApII-pSer2 normalized to DAPI. e–h Time-lapse imaging of a mouse small intestinal crypt infected with OSCAR lentivirus (movie in Supplementary Information). Upper panels correspond to fluorescent images of the brightfield images below. All the experiments were repeated at least three times with similar results. Source data are provided as a Source Data file.