Regulation of ATR Localization by PIEZO1 in Hypotonic Stress

WT or PIEZO1KO (5E3)^{43 (link)} HEK293T cells were maintained in DMEM medium supplemented with 10% FBS. Cells were transfected with pcDNA3-FLAG-ATR and GFP (pLL3.7) in the presence of Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were exposed to mock or hypotonic medium (medium diluted 1:5 with ddH2O) for 5–7 min. In some conditions, 1400 W dihydrochloride (100 μM, Santa Cruz) as a NOS inhibitor, or histamine (100 μM, Santa Cruz) as a NOS activator, were added to the hypotonic medium. For immunocytochemistry, fixed with 4% PFA for 20 min, and stained with FLAG antibody (1:500). For fluorescence intensity quantification, the integrated intensity of the whole 8-bit image was measured with ImageJ 1.52q, and the FLAG-ATR fluorescence intensity was normalized to that of GFP. For FLAG-ATR cluster analysis, DAPI was used to define the nuclear area, and the total area of FLAG-ATR clusters per cell was measured using the Analyze Particles plugin (ImageJ). To test endogenous ATR localization before and after hypotonic stress, untransfected WT and PIEZO1KO HEK293T cells were treated with hypotonic medium for 15 min before fixed with PFA. Cells were stained with ATR antibody along with the Nucleolin antibody to highlight nucleoli.