Fig. 3: Multiplex ACE profiles molecular modulations induced by differential transcriptional factor expression levels during EMT and MET processes.

a, Experimental workflow: mouse breast cancer Py2T cells were treated with TGFβ1 (4 ng ml⁻¹) for 14 days before the stimulus was withdrawn. Cells were cultured in the absence of TGFβ1 for an additional 14 days. During the timecourse, Py2T cells were harvested on the days indicated posttreatment, barcoded and pooled into a single vial for simultaneous ACE amplification and subsequent mass cytometry analysis. Experiments were performed in biological replicates to confirm reproducibility. b,c, Dimensional reduction analysis with UMAP was performed on the data. Cells are color coded by treatment time (b) or abundance of measured markers after normalization (c) on UMAP plots. d, Pseudotime analysis with Scorpius was performed and plotted against the actual time posttreatment in a violin plot. e, Signed Scorpius analysis was used to study the molecular modulation trajectories of measured markers during the EMT–MET transitioning. f, Biaxial plots show the abundances of Zeb1 and cyclin B1 levels in each single cell during the MET process across the five indicated timepoints. Dashed lines indicate the gating strategy to distinguish the Zeb1 high cyclin B1 low populations and the Zeb1 low cyclin B1 high populations. g, Boxplots showing expression levels of a mesenchymal marker vimentin and two epithelial markers, E-cadherin and CK14, in the Zeb1 high cyclin B1 low populations and the Zeb1 low cyclin B1 high populations across the five MET timepoints. Boxplots present the first quartile (Q1), median and third quartile (Q3). The interquartile range (IQR) defines distance between Q1 and Q3. The upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge. The lower whisker extends from the hinge to the smallest value no further than 1.5 × IQR of the hinge. Data beyond the end of the whiskers are plotted individually.