Figure 5.
Benzbromarone (Benz) suppresses Yoda1-induced PS exposure by inhibition of PIEZO1. (A) Benz dose-dependently inhibits Yoda1-induced PS exposure in HX001 RBCs. The flow cytometry experiments were done using CF488-AnV as a PS marker. (B) Benz dose-dependently inhibits Yoda1–induced PS exposure in HX001 RBCs. The AnV signals were normalized to zero Benz condition. The signals were fitted with the Hill equation (see “Methods”). Three independent repeats were done for each Benz concentration. (C) Representative PCLSF images of TMEM16F-mediated PS exposure in the absence (top) and presence of 20 μM extracellular Benz (bottom). Images were acquired every 5 seconds after membrane break-in under whole-cell configuration. TMEM16F was activated by pipette Ca2+. (D) Time course of TMEM16F-mediated PS exposure under PCLSF with and without Benz. t1/2 is the time for the AnV intensity to reach half maximum within the recorded time frame. The results are presented as mean ± SEM (n = 7 for each group). (E) Comparison of t1/2 with and without Benz. The results are presented as mean ± SEM. Statistical analysis was done by unpaired 2-sided Student t test (ns means no significant, n = 7 for each group). (F) Benz dose-dependently inhibits Yoda1 (2 μM)-induced Ca2+ influx in HX001 RBCs. Error bars represent SEM from 4 independent repeats. (G) Dose-response curve of Benz inhibition on Yoda1-induced Ca2+ influx in HX001 RBCs. The signals were normalized to zero Benz condition. The Ca2+ signals were fitted with the Hill equation (see “Methods”). (H) Representative cell-attached patch-clamp recording of PIEZO1 current in the presence and absence (Control) of 20 μM Benz in the pipette solution. Human PIEZO1 was overexpressed in HEK293T cells. The current was elicited by pressure clamp from 0 to −60 mmHg with a holding potential at −80 mV. (I) PIEZO1 current-pressure relationship with and without extracellular Benz. Error bars represent SEM (n = 10 and 8 for control and Benz group, respectively). (J) PIEZO1 current amplitudes at –60 mmHg with and without extracellular Benz. Statistical analysis was done by unpaired 2-sided Student t test. (∗∗∗∗P < .0001, n = 10 and 8 for control and Benz groups, respectively).

Benzbromarone (Benz) suppresses Yoda1-induced PS exposure by inhibition of PIEZO1. (A) Benz dose-dependently inhibits Yoda1-induced PS exposure in HX001 RBCs. The flow cytometry experiments were done using CF488-AnV as a PS marker. (B) Benz dose-dependently inhibits Yoda1–induced PS exposure in HX001 RBCs. The AnV signals were normalized to zero Benz condition. The signals were fitted with the Hill equation (see “Methods”). Three independent repeats were done for each Benz concentration. (C) Representative PCLSF images of TMEM16F-mediated PS exposure in the absence (top) and presence of 20 μM extracellular Benz (bottom). Images were acquired every 5 seconds after membrane break-in under whole-cell configuration. TMEM16F was activated by pipette Ca2+. (D) Time course of TMEM16F-mediated PS exposure under PCLSF with and without Benz. t1/2 is the time for the AnV intensity to reach half maximum within the recorded time frame. The results are presented as mean ± SEM (n = 7 for each group). (E) Comparison of t1/2 with and without Benz. The results are presented as mean ± SEM. Statistical analysis was done by unpaired 2-sided Student t test (ns means no significant, n = 7 for each group). (F) Benz dose-dependently inhibits Yoda1 (2 μM)-induced Ca2+ influx in HX001 RBCs. Error bars represent SEM from 4 independent repeats. (G) Dose-response curve of Benz inhibition on Yoda1-induced Ca2+ influx in HX001 RBCs. The signals were normalized to zero Benz condition. The Ca2+ signals were fitted with the Hill equation (see “Methods”). (H) Representative cell-attached patch-clamp recording of PIEZO1 current in the presence and absence (Control) of 20 μM Benz in the pipette solution. Human PIEZO1 was overexpressed in HEK293T cells. The current was elicited by pressure clamp from 0 to −60 mmHg with a holding potential at −80 mV. (I) PIEZO1 current-pressure relationship with and without extracellular Benz. Error bars represent SEM (n = 10 and 8 for control and Benz group, respectively). (J) PIEZO1 current amplitudes at –60 mmHg with and without extracellular Benz. Statistical analysis was done by unpaired 2-sided Student t test. (∗∗∗∗P < .0001, n = 10 and 8 for control and Benz groups, respectively).

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