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. 2019 Oct 9;10:1181. doi: 10.3389/fphar.2019.01181

Figure 1.

Figure 1

Inhibition of TMEM16A chloride channel activity by plumbagin. (A) Fluorescence assays in FRT-TMEM16A-YFP-H148Q/I152L cells. Left panel: Dose-dependent inhibition of TMEM16A activity by plumbagin and the structure of plumbagin. Right panel: Time-course inhibition of TMEM16A by plumbagin (n = 4). (B) MTT assay in TMEM16A-expressed FRT cells (n = 6). (C) Plumbagin inhibition of the short-circuit current induced by Eact (10 µM) in FRT cells expressing TMEM16A. T16Ainh-A01 was used as a positive control. (D) Inhibition of the short-circuit current in FRT cells in which plumbagin was added first to the basolateral bathing solution and then to the apical bathing solution as indicated, following stimulation by 10 µM Eact. (E) The blockage effect of plumbagin on TMEM16A activity. Left panel: Representative traces of short-circuit currents showing the inhibition of Eact (10 µM)-stimulated short-circuit current by 15 min of pretreatment with the indicated concentrations of plumbagin or with T16Ainh-A01 (10 µM). Right panel: Dose-response summary (n = 4). (F) Reversibility of TMEM16A inhibition by plumbagin. After incubation with 20 µM plumbagin for 15 min, the cells were stimulated with 10 µM Eact and then washed, and the TMEM16A-mediated current was reactivated by Eact. (G) Cytoplasmic calcium concentration measured by Fluo-4 NW fluorescence under basal conditions and following ATP (200 µM), CCh (100 µM), or ionomycin (1 µM) addition. HT-29 cells were pretreated with 20 µM plumbagin (n = 4).