Fig. 3. PIEZO1-mediated shear stress response in human tenocytes.

a, Mechanically-induced Ca²⁺ signals are nearly absent in Ca²⁺-free medium (containing 2 mM EGTA and 2 mM MgCl₂ instead of CaCl₂) but are restored in control medium, both in situ (one cycle stretch to 2.7% strain at 0.1% strain/s, n=7 fascicles from rat tails) and in vitro (shear stress stimulation, n=12 chambers, cells from semitendinosus tendons of 3 human donors), paired Student’s t-test. b, Highest expressed candidate genes associated with mechanosensitive ion channel characteristics selected from RNA sequencing experiments with mouse tail tendons³⁷ and human Achilles tendons³⁸. c, CRISPR/Cas9-mediated knockout efficiency of candidate genes. Normalization to gene expression in no target control cells using 2^–ddCT method (cells from n=3 human donors), significant reduction (P < 0.0001) for all candidates compared to no target control, one-way ANOVA with multiple comparisons (Dunnett’s test). d, Immunofluorescence images and Western blot analysis (full scan in Supplementary Fig. 5) showing efficient PIEZO1 knockout in human PIEZO1 knockout tenocytes compared to no target control tenocytes (scale bar, 20 μm). e-g, Ca²⁺ response of the candidate knockouts to a shear stress stimulus of 5 Pa for 5 s. PIEZO1 depleted cells show a reduced % of responsive cells and a reduced amplitude of the Ca²⁺ signals (averaged over all single segmented cells). For each candidate n≥10 chambers were tested with cells from 3 human donors (semitendinosus tendons), one-way ANOVA with multiple comparisons (Dunnett’s test). h, Two additional PIEZO1 knockouts generated with different CRISPR-guide-RNAs confirm the reduced shear stress response (n≥4 chambers, cells from a human semitendinosus tendon), one-way ANOVA with multiple comparisons (Dunnett’s test). Replicates are biological. Data are means±SEM.