Figure 3.

Inhibition of endogenous Gli1 and Gli2 activity results in an increase ANO1 channel activity. A) Effect of inhibition of endogenous Gli function on ANO1 RNA expression in HEK293 cells. Quantitative RT-PCR data are shown as fold changes over untreated cells. Data are expressed as the mean ± se of 3 independent experiments (n = 3). *P < 0.05, 2-way ANOVA with Bonferroni’s multiple comparison test. B) Representative confocal images of HEK293 cells immunostaining using an anti-ANO1 antibody (red) after treatment with either vehicle or GANT61 compound for 48 h. In blue is the nuclear counterstain with DAPI. Scale bar, 20 µm. C) Representative traces of functional Ca2⁺-activated C^l− channels recorded from HEK293 cells treated with vehicle (black) or GANT61 (20 µM, red). Cells were assayed by whole cell voltage clamp recordings optimized for ANO1. D) Quantification of voltage clamp data recorded from HEK293 cells treated with vehicle (black) or GANT61 (red) represented as current (I)-voltage (V) plots (n = 6–13 cells). *P < 0.05, 2-tailed Student’s t test. E) Effect of asymmetric chloride ratio on measured chloride currents after treatment of HEK293 cells with GANT61 (red) or vehicle (black). On the y axis is indicated the measured reversal potential for chloride in mV (E_REV). The chloride ratio between extracellular ([Cl]_o) and intracellular ([Cl]_i) solutions is plotted on the x axis (n = 6–13). *P < 0.05, 2-tailed Student’s t test. ASYM, asymmetrical chloride distribution; SYM, symmetrical chloride distribution. F) Effect of the inhibitor T16Ainh-A01 (30 µM) on measured chloride currents from HEK293 cells treated with GANT61. The currents measured after GANT61 treatment are plotted on the y axis; the voltage (mV) are plotted on the x axis (n = 4). *P < 0.05, 2-tailed Student’s t test.