Studies of the E2496ELE PIEZO1 mutant. (A) HEK293 cells expressing the E2496ELE mutant displayed morphological changes upon induction. Top panels: compared with wild type, the mutant cells demonstrated swelling as early as 16 hours postinduction with rounding up of edges, followed by wide-scale detachment of cells from the plate by 48 hours. Bottom panels: none of the known PIEZO1 inhibitors, including the G spatulata mechanotoxin (GsMTx4), gadolinium, or ruthenium red, protected cells from this effect. Original magnification ×100. (B) Live cell numbers and annexin V staining. Beginning at ∼48 hours postinduction, the number of live, mutant cell numbers determined by CCK8 staining decreased dramatically (left graph) and cells began to undergo apoptosis as assayed by annexin V staining (right). Camptothecin (CPT) was used for artificial induction of apoptosis in wild-type cells. Scale bars, 1 μm. FITC, fluorescein isothiocyanate.