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. 2020 Dec 21;153(2):e202012704. doi: 10.1085/jgp.202012704

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© 2020 Liang and Yang

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Figure 4. — pH_i regulation of TMEM16F scrambling activity is Ca²⁺ dependent. (A) Representative images of TMEM16F-eGFP scrambling activity under 5 µM intracellular Ca²⁺ with different pH_i values. The white dotted rectangles in the top row demarcate the patch-clamped TMEM16F-eGFP–expressing cells. For the AnV-CF 594 signals on the right, the first column is fluorescence signal immediately after forming whole-cell configuration; the second column is the time point (minutes followed by seconds, top right corner) when fluorescence intensity reaches half maximum (t_1/2), and the last column is the time point when fluorescence reaches plateau. No obvious AnV-CF 594 signal can be detected in pH_i 6.1 over 40 min. The recording interval is 10 s. See also Video 3. (B) The time courses of AnV fluorescence intensity for TMEM16F activated by 5 µM Ca²⁺ under different pH_i values in A. The smooth curves represent fits to the logistic equation similar to Fig. 2 B. (C) The scrambling onset time (t_on) at different pH_i values under 5 µM Ca²⁺. N/S at pH_i = 6.1 represents no scrambling. Error bars represent SEM (n = 5). (D) The t_1/2 at different pH_i values under 5 µM Ca²⁺. N/S at pH_i = 6.1 represents no scrambling. Error bars represent SEM (n = 5). (E) The slopes at different pH_i values under 5 µM Ca²⁺. (F) Representative images of TMEM16F-eGFP scrambling activity under 1,000 µM intracellular Ca²⁺ with different pH_i. For the AnV-CF 594 signal on the right, the first column is fluorescence signal immediately after forming whole-cell configuration; the second column is the time point when fluorescence intensity reaches half maximum (t_1/2), and the last column is the time point when fluorescence reaches plateau. See also Video 4. (G) Time courses of AnV fluorescence intensity for TMEM16F activated by 1,000 µM Ca²⁺ under diff,erent pH_i values in F. The smooth curves represent fits to the logistic equation. (H) The scrambling onset time (t_on) at different pH_i values under 1,000 µM Ca²⁺. Error bars represent SEM (n = 5). (I) The t_1/2 of lipid scrambling at different pH_i values under 1,000 µM Ca²⁺. (J) The slopes (k) at different pH_i values under 1,000 µM Ca²⁺. Error bars represent SEM (n = 5). Statistics were analyzed using one-way ANOVA followed by Tukey’s test. **, P < 0.01; ***, P < 0.001; ns (not significant), P > 0.05.