FIGURE 3.

Effect of Piezo1 inhibitors on F-actin organization. (A) Images of F-actin reorganization in the presence of GsMTx4 and Gd³⁺, showing the stress fibers disassembled in the presence of inhibitors (yellow arrows indicate stress fibers, blue arrows indicate peripheral actin rings). The inhibitors were added during cell seeding, and the cells were fixed and stained after 2.5 h. Scale bars represent 20 µm. (B) Statistical analysis of interior actin intensity with and without GsMTx4, which shows that blocking Piezo1 channels reduced the actin intensity on stiffer substrates (n = 50 for all conditions, *p < 0.001). (C) Comparison of spreading areas on hard and soft substrates in the presence of inhibitors, showing that blocking Piezo1 channels did not drastically alter cell spreading (n = 50 for all conditions, *p < 0.005). (D) F-actin thickness with and without inhibitor, showing that blocking Piezo1 channels reduced the thickness of fibers significantly (n = 170/70; 220/50; 60/60 for control/drug on glass, hard, soft, respectively, measured from 30 cells in each condition, *p < 0.001). (E) Number of actin fibers per cell (n = 30 cells, *p < 0.001).