Figure 2.

HSP90 inhibition reduces T2R-stimulated NO diffusion into the airway surface liquid (ASL) in H441 cells grown at the air–liquid interface (ALI) (A): Representative images and bar graph of 4 independent experiments of fluorescence at the apical plane of ALI when 100 µL of solution containing cell impermeable DAF-2 was placed on top (1.1 cm² Transwell) either containing sodium benzoate (top) or denatonium benzoate (bottom). Cultures were either pretreated with 0.1% DMSO (vehicle control), 10 µM PLC inhibitor U73122, or 10 µM inactive analogue U73343 prior to the experiment. Significance determined by one-way ANOVA with Dunnett’s post-test comparing all values to HBSS only control. (B): Bar graph of experiments performed as in (A) but testing inhibition of denatonium-induced or quinine-induced ASL DAF-2 fluorescence ± NOS inhibitor L-NAME or inactive D-NAME (10 µM). Bar graph shows the mean ± SEM of 3–5 independent experiments imaged at identical conditions. Significance by one-way ANOVA with Bonferroni post-test comparing all values to respective HBSS control; ** p < 0.01. (C): Denatonium-stimulated H441 DAF-2 ASL fluorescence increases were reduced in the presence of GPCR signaling inhibitor YM254890 or HSP90 inhibitors geldanamycin, 17-AAG, or BIIB 021. HSP70 inhibitor VER-15508 had no effect. Bar graph shows the mean ± SEM of four independent experiments. Significance by one-way ANOVA with Dunnett’s post-test comparing all values to control (0.1% DMSO only); * p < 0.05 and ** p < 0.01. (D): H441s were treated with siRNA as described in the methods. ASL DAF-2 responses during denatonium stimulation were reduced by eNOS siRNA but not with scramble, nNOS, or PAR-2 siRNA. Bar graph shows the mean ± SEM of four independent experiments (separate siRNA transfections). Significance by one-way ANOVA with Dunnett’s post-test comparing all values to control; ** p < 0.01.