Fig. 5: Spatial-temporal quantification of mRNAs and proteins after LIF withdrawal from mESCs on micropatterns.

a Schematic of the experiment where ARTseq-FISH was performed at 0, 12, 24 and 48 h after LIF withdrawal. Created with BioRender.com. b Schematic of the definition of centre and edge on the micropattern. Created with BioRender.com. c Heatmap showing the abundance of different classes of targets (see Supplementary Data 5 for full list of targets) across 1 micropattern at 0, 12, 24 and 48 h after LIF withdrawal. The expression level of targets is normalised to the maximum abundance across all four time points. d Relative single-cell expression levels of the targets shown in (c), at the edge (top) and the centre (bottom) of the micropattern at 0, 12, 24 and 48 h after LIF withdrawal. 0–24-h data includes two biological replicates; 48 h includes three biological replicates (for the edge 393 cells and the centre 238 cells were analysed in total). For each replicate and time point, 1 micropattern was analysed. The line represents the mean. Shaded regions represent 95% confidence intervals. e Single cell NANOG protein expression at the edge and centre of micropattern at 0, 12, 24 and 48 h after LIF withdrawal detected by ARTseq-FISH (left). 0–24-h data includes two biological replicates; 48 h includes three biological replicates (edge and centre include 393 and 238 cells in total, respectively). For each replicate and time point, 1 micropattern was analysed. Quantification of live cell time-lapse of NANOG-GFP positive mESCs for five different positions at the edge and the centre of 1 micropattern over 46.5 h of LIF withdrawal (right). The line represents the mean. Shaded regions represent 95% confidence intervals. f Pearson correlation and hierarchical clustering of pluripotency and lineage-related markers at the edge of the micropattern (3 biological replicates, 1 micropattern each, 111 cells total) at 48 h after LIF withdrawal. g (i) UMAP clustering of expression levels of 57 markers of individual cells within a single micropattern experiment at different time points (0, 12, 24 and 48 h) after LIF withdrawal. (ii) UMAP clustering of expression levels of 57 markers of single cells at different positions (centre, edge or between centre and edge) within the micropattern (purple, orange and white, respectively). One biological replicate and 1 micropattern (0, 12, 24 and 48 h include 269, 139, 141 and 158 cells, respectively). Source data are provided as Source Data sheet tabs Fig. 5d–g.