Fig. 4: Functional validation of the drug binding site in TMEM16F. | Nature Communications

Fig. 4: Functional validation of the drug binding site in TMEM16F.

From: Identification of a drug binding pocket in TMEM16F calcium-activated ion channel and lipid scramblase

Fig. 4

Representative curves of live imaging of TMEM16F-dependent PS exposure (a–d); and Ca2+ influx (e–h). NC is negative control with stable cell line expressing mScarlet rather than TMEM16F tagged with mScarlet. Data are represented as mean ± SEM. Scattered dot plots of time of onset of TMEM16F-dependent PS exposure (i) n = 76 for wt Ctrl; 14 for wt Niclo 1 μM; 29 for wt Niclo 3 μM; 25 for wt Niclo 10 μM; 28 for wt 1PBC 1 μM; 25 for wt 1PBC 3 μM; 29 for wt 1PBC 10 μM; n = 23 for Triple Ctrl; 25 for Triple Niclo 1 μM; 22 for Triple Niclo 3μM; 30 for Triple Niclo 10 μM; 28 for Triple 1PBC 1 μM; 25 for Triple 1PBC 3 μM; 42 for Triple 1PBC 10 μM and Ca2+ influx (j) n = 31 for wt Ctrl; 15 for wt Niclo 1 μM; 12 for wt Niclo 3 μM; 9 for wt Niclo 10 μM; 22 for wt 1PBC 1 μM; 31 for wt 1PBC 3 μM; 15 for wt 1PBC 10 μM; n = 24 for Triple Ctrl; 11 for Triple Niclo 1 μM; 13 for Triple Niclo 3 μM; 6 for Triple Niclo 10 μM; 28 for Triple 1PBC 1 μM; 23 for Triple 1PBC 3 μM; 17 for Triple 1PBC 10 μM. Time of onset could not be determined for time courses with a linear rather than sigmoidal rise. At least three independent experiments have been performed for each condition, each with distinct cell populations to assess biological rather than technical variability. The mean ± SEM is shown along with the statistical significance determined by unpaired t-test (two-tailed) for wildtype or mutant with 1PBC or Niclo as compared to its vehicle control (wt Niclo 1 μM: p < 0.0001; wt Niclo 3 μM: p < 0.0001; wt Niclo 10 μM: p < 0.0001; wt 1PBC 1 μM: p < 0.0001; wt 1PBC 3 μM: p < 0.0001; wt 1PBC 10 μM: p < 0.0001; Triple Niclo 1 μM: p = 0.8613; Triple Niclo 3 μM: p = 0.3651; Triple Niclo 10 μM: p = 0.1865; Triple 1PBC 1 μM: p < 0.0001; Triple 1PBC 3 μM: p = 0.1166; Triple 1PBC 10 μM: p = 0.3885 in (i) and wt Niclo 1 μM: p = 0.0004; wt Niclo 3 μM: p < 0.0001; wt Niclo 10 μM: p < 0.0001; wt 1PBC 1 μM: p = 0.3469; wt 1PBC 3 μM: p = 0.0003; wt 1PBC 10 μM: p < 0.0001; Triple Niclo 1 μM: p < 0.0001; Triple Niclo 3 μM: p < 0.0001; Triple Niclo 10 μM: p < 0.0001; Triple 1PBC 1 μM: p = 0.8231; Triple 1PBC 3 μM: p = 0.0161; Triple 1PBC 10 μM: p < 0.0001 (j), ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). Triple: T606A/T607A/T610A.

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