Fig. 4: ARTseq-FISH reveals gradients of mRNA and protein expression levels within micropattern-differentiated mESCs.

a Schematic illustration of three representative spatial distributions of molecules on micropatterns. Created with BioRender.com. b Immunofluorescence results of p53, Phos-Pol II (Phosphorylated RNA Pol II), and BRACHYURY within a micropattern-differentiated mESCs. Scale bar, 100 µm. c Average number of spots per cell at different positions on the micropattern (edge to centre) for p53, Phos-Pol II and BRACHYURY proteins from three biological replicates (150, 125 and 158 cells and 1 micropattern were per replicate). d Pearson correlation between relative mean fluorescence in (b) and relative spots per cell with respect to their position on the micropattern (c). e Mean fold change of the expression level of individual targets between the edge and centre (purple and orange respectively) of the micropattern across four biological replicates (125–158 cells and 1 micropattern analysed per replicate). *: phosphorylated protein. Error bars represent the SEM of four biological replicates. f UMAP analysis of expression levels of 57 markers of individual cells after 48 h of LIF withdrawal, with the colour representing if cells are localised towards the edge (orange) or centre (purple) of the micropattern (left). The same UMAP with single cell abundance of p53, Phos-Pol II and BRACHYURY proteins indicated in blue. 158 cells and 1 micropattern were analysed. g The spots per cell (y-axis) with respect to the position of the cell on the micropattern (x-axis) of selected targets. The edge of the micropattern serves as the reference value for the relative position of the cells. Cells are found at the edge at 0 µm and near the centre at 375 µm. 158 cells were analysed per target. Source data are provided as Source Data sheet tabs Fig. 4c–g.