Fig. 2: NHE9 drives Ca²⁺-mediated epithelial-mesenchymal plasticity.

a Schematic depiction of the NHE9-coupled EMT circuit. The core regulatory network is boxed. Dotted line represents micro-RNA mediated regulation. The correlation coefficients between NHE9 expression and different regulators in the EMT circuit derived from CRC samples in the TCGA database are displayed. Scatter plots are shown in Supplementary Fig. 2. b Mathematical model predicts that NHE9 promotes acquisition of a hybrid E/M state. Bifurcation diagrams comparing ZEB mRNA levels for increasing EMT-inducing signal (SNAIL levels) in the NHE9-coupled EMT circuit (solid blue and dotted red curve) to the control or core EMT circuit (solid green and dotted black curve). Note that continuous curves represent stable steady-state solutions, while dotted lines represent unstable solutions (left). Temporal dynamics of ZEB mRNA levels in an epithelial cell exposed to a high level of EMT-inducing signal (SNAIL) for core (blue) and NHE9-coupled (green) EMT circuits. The numbers 1 and 2 denote hybrid E/M regions of the core and NHE9-coupled EMT circuits, respectively (right). c Scatter plots depicting the relationship of NHE9 with ZEB1 (top) and ZEB2 (bottom) mRNA in CRC samples from the TCGA database (n = 592). Each plot shows the linear fit and 95% confidence interval, Pearson’s correlation coefficient (R), and P-value. d Bar graph depicting frequencies of different stable phases for the NHE9-coupled EMT circuit compared to the control EMT circuit obtained across parameter sets generated for three independent RACIPE replicates. The error bars reflect standard deviation. See text for details. e Scatter plot depicting the association between NHE9 and ZEB1 levels in the minimal model calculated from RACIPE analysis. f Schematic of the experimental protocol after transduction with the indicated plasmids. g Plots of epithelial signature (EPI) and mesenchymal signature (MES) scores of colorectal cancer cells with NHE9 expression versus EV control. Lower EPI scores and higher MES score upon NHE9 expression indicate a loss of epithelial nature and a gain of mesenchymal status. h Scatter plots depicting the relationship between [Ca²⁺] signature scores (as measured by ssGSEA) and EPI and MES signatures. Each plot shows the linear fit and 95% confidence interval, Pearson’s correlation coefficient (R), and P-value. The illustrations were created with BioRender. P values were calculated by Pearson’s correlation (c, e, h) and unpaired, two-tailed t test (d, g). See related Supplementary Figs. 2, 3.