Journal: bioRxiv

Article Title: Log-linear scaling of TRPV4-KCNN4 transcripts tunes ROCK-dependent mechanotransduction in a DCIS progression model

doi: 10.64898/2026.02.19.706850

Figure Lengend Snippet: (A) Heatmap of candidate mechanotransduction genes across six cell lines ordered by increasing mechanotransduction capacity (ETCC-06 lowest to MCF10DCIS.com highest). Values are log2(mRNA_cell line/mRNA_MCF10DCIS.com). NA indicates log2 fold-change values that were not available from the differential-expression outputs used for fold-change extraction, resulting in gene-specific n<6 for some genes (e.g., PIEZO1/2). Superscripts indicate predominant localization: M, membrane; C, cytosolic; N, nuclear. (B) Summary correlations between candidate gene expression (log2FC vs MCF10DCIS.com) and mechanotransduction output (MI_PEG). Bars denote expression dynamic range across the panel; overlaid points report R² from linear regression of gene expression versus MI_PEG (red, p<0.05; orange, p≥0.05; purple, significant inverse correlation (p<0.05)). Among surveyed candidates, TRPV4 (R²=0.92, p=0.003) and KCNN4 (R²=0.94, p=0.002) show the strongest positive predictive relationships. (C) KCNN4 mRNA abundance predicts MI_PEG with log-linear scaling. Points represent individual cell lines; error bars: mean±SD. Dashed line: full panel regression (n=6, R²=0.94, p=0.002); solid line: isogenic MCF10A series (n=4, R²=0.81, p=0.099). (D) KCNN4 mRNA abundance predicts MI_GSK219 with similar log-linear scaling. Same plotting conventions as in (C) . Dashed line: full panel (n=6, R²=0.81, p=0.015); solid line: isogenic series (n=4, R²=0.67, p=0.184). (E) TRPV4 and KCNN4 mRNA levels are strongly correlated across the cell-line panel, indicating coordinated transcriptional regulation. Dashed line: full panel (n=6, R²=0.83, p=0.012); solid line: isogenic MCF10A series (n=4, R²=0.90, p=0.050). (F) Functional validation: pharmacologic KCNN4 inhibition (TRAM-34, 5 μM) phenocopies the motility increase induced by hyperosmotic stress (PEG300; Δ74.4 mOsm/L). Distributions show single-cell diffusion coefficients ( D ) for control, TRAM-34, PEG, and PEG+TRAM-34. TRAM-34 (0.094±0.120 µm²/s) and PEG (0.120±0.111 µm²/s) each increased motility versus control (0.054±0.088 µm²/s; both p<0.0001), with no additive effect in the combined condition (PEG+TRAM-34: 0.099±0.124 µm²/s, ns vs either alone). Each point is one cell; >100 cells total were tracked per condition across two independent experiments. Statistics: Mann-Whitney U test. **p<0.01; ****p<0.0001; ns, not significant.

Article Snippet: During this incubation, cells received the following treatments in complete media: 1 nM GSK2193874 (GSK219, MedChemExpress HY-100720) for 1 hr, 5 μM TRAM-34 (MedChemExpress HY-13519) for 1 hr, 50 μM Y-27632 dihydrochloride (ROCK Inhibitor, Sigma Aldrich Y0503) for 1 hr, or Δ74.4 mOsm/L Polyethylene glycol 300 (PEG300, Millipore Sigma 8074845000) for 15 min. For combination treatments, when multiple treatments were performed within the same dish, the first treatment was applied for its full incubation time before addition of the second treatment: order of addition was as follows: 5 μM TRAM-34 followed by Δ74.4 mOsm/L PEG300, 50 μM ROCK Inhibitor followed by Δ74.4 mOsm/L PEG300, 1 nM GSK219 followed by Δ74.4 mOsm/L PEG300, or 1 nM GSK219 followed by 5 μM TRAM-34.

Techniques: Quantitative Proteomics, Extraction, Membrane, Gene Expression, Expressing, Functional Assay, Biomarker Discovery, Inhibition, Single Cell, Diffusion-based Assay, Control, MANN-WHITNEY