(A) Fraction of chondrocytes that responded with at least with one mechanically gated current in response to deflection stimuli. Knockdown of Piezo1 resulted in significantly fewer responsive cells compared with cells treated with non-targeting miRNA (scrambled) (Fisher´s exact test, *p=0.04). Trpv4-/- chondrocytes were significantly less likely to respond to deflection stimuli compared with WT cells (Fisher’s exact test, *p=0.03). When the miRNA against Piezo1 was expressed in Trpv4-/- chondrocytes, the response further decreased compared with the WT chondrocytes transfected with a scrambled miRNA (***p=0.002, Fisher’s exact test). (B) Stimulus-response graph of the deflection-gated currents in chondrocytes transfected with a scrambled miRNA (gray open circles, n = 22 cells) or Piezo1-targeting miRNA (green open circles, n = 12 cells). Data are displayed as mean ± s.e.m., and a representative trace of the mechanosensitive currents is shown as insert (green line). (C) Cells isolated from a Trpv4-/- mouse are significantly less sensitive to deflections, in comparison with WT cells. Stimulus-response graph of the mechanically gated currents triggered by pillar deflections in WT chondrocytes (black open circles, n = 27 cells) and Trpv4-/- chondrocytes (magenta open circles, n = 13 cells). The Trpv4-/- cells are significantly less responsive to substrate deflections (ordinary two-way ANOVA, *p=0.04). Data are displayed as mean ± s.e.m. A representative trace is shown as insert (magenta line). (D) Stimulus-response graph of Trpv4-/- chondrocytes transfected with a Piezo1-targeting miRNA. Data are displayed as mean ± s.e.m. (n = 11 cells). Chondrocytes from the Trpv4-/- mouse treated with Piezo1-targeting miRNA were significantly less sensitive to substrate deflections, in comparison with WT cells treated with scrambled miRNA (ordinary two-way ANOVA, *p=0.04). A representative trace is shown as insert (black line). (E) Flourometric calcium imaging of chondrocyte responses to Yoda1 and GSK1016790A. Cells were perfused with ATP (10 µM), Yoda1(10 µM) and GSK1016790A (GSK101, 50 nM) as indicated by black bars and changes in [Ca2+] were monitored by using the Ca2+ responsive dye, Cal520. In the left panel, traces correspond to intensity changes in individual cells and in the right panel is a plot representing the average of all cells (as mean ± s.e.m.). Example images are presented of cells before activation, during application of Yoda1 and of GSK1016790A. Scale bar 20 µm. Each cell that responded to ATP was included in the analysis (400 cells, two preparations).