TMEM16A chloride channel does not drive mucus production

(A) Confocal immunofluorescence microscopy images showing TMEM16A localization in permeabilised BCi-NS1.1 cells on days 0 and 30 (upper and lower rows, respectively) of differentiation. Left panels: Nuclei stained with Hoechst stain. Middle panels: endogenous TMEM16A, detected by Alexa Fluor 568 fluorescence. Endogenous ZO-1 was detected by Alexa Fluor 488 fluorescence. Right panels: merged image of the three fluorescent channels: Blue, Alexa 647; Red, Alexa 568; and Green, Alexa 488. Images were acquired with a Leica TCS SP8 confocal microscope (objective 63× oil, NA 1.4). Scale bar = 30 μm. (n = 3). (B) Z-stack of a representative group of cells showing TMEM16A and ZO-1 staining on days 0 and 30 (upper and lower panels, respectively). (C) Confocal immunofluorescence microscopy images showing MUC5AC expression in permeabilised BCi-NS1.1 cells on days 0 and 30 of differentiation (upper and lower rows, respectively). Left panels: nuclei stained with metal green, represented by Alexa Fluor 647 fluorescence. Middle panels: endogenous MUC5AC, detected by Alexa Fluor 488 fluorescence. Right panels: merged image of the two fluorescent channels: Green–Alexa 488; Blue–Hoechst. Images were acquired with a Leica TCS SP8 confocal microscope (objective 63× oil, NA 1.4). Scale bar = 30 μm. (n = 3). (D) Z-stack of a representative group of cells showing MUC5AC stained apically on days 0 and 30 (upper and lower panels, respectively).

(A) Time course levels of endogenous expression of Ki-67 protein (∼300 kD) during differentiation of BCi-NS1.1 grown at ALI (days 0-30) analysed by WB. GAPDH (∼36 kD) was used as loading control. (B) Quantification by densitometry of Ki-67 expression detected by WB and normalized to the loading control shown as mean ± SEM (n = 3). (C) Correlation of TMEM16A and Ki-67 normalized protein levels and TEER measurements during differentiation. Summary of TMEM16A total protein levels represents data in Fig 1. (D) Time course (0 h to 5 d) of expression levels of endogenous expression of TMEM16A and Ki-67 during wound closure (0, 8, 24, 48 h, and 5 d after injury) on the 20th day of differentiation at ALI analysed by WB. GAPDH (∼36 kD) was used as loading control. (E) Quantification by densitometry of total TMEM16A and Ki-67 expression detected by WB and normalized to the loading control shown as mean ± SEM (n = 3–5). Asterisks indicate significant difference compared with day 0 (in graph (B)) or before wound (in graph (E)) (P-value < 0.05, unpaired t test).

Source data are available for this figure.

(A) WB of endogenous TMEM16A and Ki-67 proteins during differentiation of primary HBE cells. GAPDH was used as loading control. (B) Quantification by densitometry of total TMEM16A and Ki-67 expression detected by WB and normalized to the loading control shown as mean ± SEM (n = 3). (C) WB of endogenous MUC5AC during differentiation of primary HBE cells. Vinculin was used as loading control. (D) Quantification by densitometry of MUC5AC detected by WB and normalized to the loading control shown as mean ± SEM (n = 3). Asterisks indicate significant difference compared with day 0 (P-value < 0.05, unpaired t test).

Source data are available for this figure.

(A) WB indicating up-regulation of TMEM16A, MUC5AC, and Ki-67 by stimulation with 5 ng/ml IL-4 for 48 h in BCi-NS1.1 cells. GAPDH was used as a loading control for TMEM16A and Ki-67 WB. Vinculin was used as a loading control for MUC5AC WB. (B) Quantification by densitometry of WB for total TMEM16A, MUC5AC, and Ki-67 expression normalized to the loading control, data shown as mean ± SEM (n = 3). (C) Original Ussing chamber tracings ± IL-4, obtained for ATP-induced Cl⁻ currents (100 μM) in the presence of the epithelial Na⁺ channel (ENaC) inhibitor, amiloride (30 μM). Reduction of the ATP-activated Cl⁻ currents was observed under the CaCC-AO1 TMEM16A inhibitor (30 μM). (D) Summary of Isc-eq ATP currents in the presence or absence of IL-4. Values are mean ± SEM (n = 3–7). Asterisk indicates significant difference compared with control; cardinal indicates significant difference compared with ATP alone (P-value < 0.05, unpaired t test). (E) WB showing down-regulation of TMEM16A and Ki-67 expression by treatment with the proliferation blocker mitomycin C (1 μg/ml) and IL-4 (5 ng/μl) for 48 h. Na⁺/K⁺ ATPase (∼100 kD)—another protein expressed in the membrane—was detected as a control. GAPDH was used as loading control. Dashed line indicates lanes run on the same gel but noncontiguous. (F) Quantification by densitometry of total TMEM16A, Ki-67, and Na⁺/K⁺ ATPase expression detected by WB and normalized to the loading control, shown as mean ± SEM (n = 5–7). (G) WB showing MUC5AC levels upon treatment with mitomycin C (1 μg/ml) ± IL-4 (5 ng/ml) for 48 h. Vinculin was used as loading control. Dashed line indicates lanes run on the same gel but noncontiguous. (H) Quantification by densitometry of MUC5AC detected by WB and normalized to the loading control, shown as mean ± SEM (n = 4–5). Asterisks indicate significant difference compared with control; cardinals indicate significant difference compared with treatment with IL-4 (P-value < 0.05, unpaired t test).

Source data are available for this figure.

(A) WB of endogenous TMEM16A and Ki-67 expression in the presence or absence of 400 ng/μl DLL4 for 30 d. Samples from undifferentiated cells were used as a positive control for TMEM16A and Ki-67 expression. GAPDH was used as loading control. (B) Quantification by densitometry of total TMEM16A and Ki-67 expression detected by WB and normalized to the loading control shown as mean ± SEM (n = 3). (C) WB of endogenous MUC5AC in the presence or absence of 400 ng/μl DLL4. Vinculin was used as a loading control. (D) Quantification by densitometry of MUC5AC expression detected by WB and normalized to the loading control shown mean ± SEM (n = 3). Asterisks indicate significant difference compared with control (P-value < 0.05, unpaired t test).

Source data are available for this figure.

(A) Representative confocal images of ASL labelled with FITC-Dextran obtained 0.5 and 4 h after apical exposure to DMSO or 10 μm Ani9. (B) Quantification of overtime (0.5, 1, 2, 3, and 4 h) ASL height measurements shown as mean ± SEM (n = 7–9). Asterisks indicate significant difference compared with 0.5 h (P-value < 0.05, unpaired t test).