[8/13] To move from this catalog of mechanosensitive sites to causal candidates, we applied a two-step filter: first, we retained phosphosites with documented regulatory function in curated databases; then, within the transcription factor category, we kept only those phosphorylated at residues with defined roles in transcriptional control. Six high-confidence mechanosensitive transcription factors remained: EP300, TP53, NFATC4, FOXO3, YBX1, and RB1.

[9/13] NFATC4 stood out to us: Using transcription factor regulon analysis of a multicohort scRNA-seq atlas of pulmonary fibrosis, we found that NFATC4 activity is elevated in IPF fibroblasts compared with healthy controls. Its inferred regulon in IPF fibroblasts placed CTHRC1 and COL1A1 among its top induced targets, tying it directly to the CTHRC1+/ACTA2+ state that defines pathological fibrosis across several organs. Across independent scRNA-seq studies, CTHRC1 has emerged as the defining marker of the pathological fibroblast population that expands in fibrotic lung, heart, and liver — a transcriptionally distinct, high collagen–producing subset largely absent from healthy tissue.

[10/13] Immunofluorescence analysis of micro-CT staged IPF lung tissue showed NFATC4, CTHRC1, and ACTA2 increasingly colocalized as disease severity rose. This gradient is what one would expect if NFATC4-mediated mechanosensing is engaged progressively as tissue stiffens, and it anchors the S213/S217 axis as a disease-relevant regulatory node in the human lung.

[11/13] NFATC4 siRNA in primary lung fibroblasts from healthy donors as well as IPF donors reduced ECM secretion and myofibroblast marker expression including CTHRC1. Interestingly, we could also show using fibronectin coated adhesive micropatterns that knock down cells displayed reduced contractility and nuclear deformation phenotypes, suggesting that the contractility enhancing reinforcement feedback that engages during cellular mechanosensing depends on NFATC4.

[12/13] Finally, using site-specific phosphomimetic (S213D/S217D) and phospho-dead (S213A/S217A) mutants, we showed that phosphorylation at these two residues is both necessary and sufficient to drive fibroblasts into the CTHRC1+/ACTA2+ state. The phosphomimetic mutant forced differentiation even on soft (0.5 kPa) substrates and without TGF-β — conditions under which control fibroblasts stay quiescent — whereas the phospho-dead mutant was unable to activate myofibroblast markers even on stiff matrix in the presence of TGF-β. Critically, overexpressing WT NFATC4 at comparable levels did nothing under any condition, establishing that fate is set by the phosphorylation status of NFATC4 rather than its abundance. This mirrors the well-established logic of the NFAT family, whose activity is gated by phosphorylation-dependent localization rather than expression level, and it identifies posttranslational control as the decisive layer linking mechanical input to cell identity. Pharmacological JNK inhibition phenocopied the phospho-dead mutant in healthy and IPF patient–derived fibroblasts, consistent with JNK acting upstream of the NFATC4 phospho-switch and showing that the mechanism is conserved across healthy and already activated fibrotic cells.