(A) Flow cytometry histograms of Fluo-4 fluorescence of WT (left) or Vav1-P1cKO (right) RBCs treated with vehicle (gray shaded), 15 μM Yoda1 (solid line), or 10 μM A23187 (dashed line) for 1 min as indicated. Rightward shifts in fluorescence indicate increased intracellular Ca2+. (B) Brightfield images from RBCs from WT (top) or Vav1-P1cKO (bottom) RBCs at the indicated times after superfusion with 15 μM Yoda1. (C) Osmotic fragility (±SEM, n = 3) of blood from WT (left) or Vav1-P1cKO (middle) treated with 2 μM the KCa3.1 antagonist TRAM-34 and/or 5 μM Yoda1 as indicated. Blood was incubated with TRAM-34 or vehicle for 10 min, and then incubated with Yoda1 or vehicle for 30 min. Graph on right depicts C50 ± SEM for hemolysis for the genotypes and treatments in the left graphs. p values were calculated using one-way ANOVA. (D) Osmotic fragility (±SEM, n = 3) of WT and Vav1-P1cKO blood treated with 1 μM of the Ca2+ ionophore A23187 for 30 min. *p < 0.05; ***p < 0.001 compared to genotype-matched, vehicle-treated blood by Student's t-test. (E) Working model for how Piezo1 activation regulates RBC volume. Experiments were repeated the following number of times: A: 3, B: 3, C: 2, D: 2, with results from an individual experiment being presented.