NC cells were cultured on fibronectin-coated coverslips and fixed in 3.7% formaldehyde in PBS for 30 min. Permeabilization was performed with 0.1% Triton X-100 in PBS for 10 min followed by PBS-Tween 0.1% washes. Primary antibodies were incubated for 16 h at 4°C. The following primary antibodies were used: anti-Piezo1 (1:500, ab128245, Abcam); anti-p-paxillin (1:500, pY118, Invitrogen); anti-vinculin (1:300, V9131, Sigma-Aldrich); anti-Rac1-GTP (1:500, sc-514583, Santa Cruz Biotechnology). Alexa fluor Phalloidin (1:500, A12379, Thermo Fisher Scientific); Alexa fluor secondary antibodies (1:500, A11008, A11001, Invitrogen) and Dapi counterstain (20 μg/ml, D9542, Sigma-Aldrich) were incubated for 30 min at 25°C. Imaging was carried out using a Leica TCS SP8 confocal with a 40× lens (HC PL APO 40×/1.30 Oil CS2).
For each experiment, the control and treated samples were manipulated in parallel and following the same staining/imaging conditions. For fluorescence intensity, a background noise subtraction was performed before intensity measurements. The intensity values for each experiment were normalized to the mean of the control.
For each experiment, the control and treated samples were manipulated in parallel and following the same staining/imaging conditions. For fluorescence intensity, a background noise subtraction was performed before intensity measurements. The intensity values for each experiment were normalized to the mean of the control.