(A) A representative trace of the fast component. The waveform of the pressure pulse is shown above the trace. (B) The fast component is attenuated by non-selective Piezo1 antagonists ruthenium red (RR, 10 μM) and GsMTx4 (5 μM), but not a TRPV4 antagonist HC06047 (5 μM). Shown are representative traces. (C) Quantification of results illustrated in B. Shown is the mean ± SD values of the fast response. The response was calculated by subtracting the baseline current from the peak current. N.S.p = 0.7252; ###p = 0.00012 and p = 0.0010 for control vs. HC067047, contro vs. RR, and control vs. GsMTx4, respectively. Two-sample t-test. N = 2 eyes, n = 15, n = 7, n = 15, and n = 9 cells for untreated (control) cells and cells treated with HC067047, RR and GsMTx4, respectively. (D) Validation of Piezo1 shRNA constructs by Q-PCR analysis. Shown are the mean ± SD. N = 3 independent experiments. The insert is a representative fluorescent image of a TM cell overexpressing Piezo1 shRNA-mCherry construct. (E) The fast component is attenuated by in cells overexpressing Piezo1 shRNA but not sc shRNA. Shown are representative traces. (F) Bar graphs summarizing effects of Piezo1 knockdown on the fast component of pressure response. The response was calculated by subtracting the baseline current from the peak current. Shown are the means ± SD. ##p = 0.0031, two-sample t-test. n = 4 cells and n = 4 cells for scrambled control (Sc-shRNA) and Piezo1-shRNA overexpressing cells, respectively. The holding potential in A-C and E and F was −40 mV.