Inhibition of endogenous PLC prevented the alpha-toxin-induced release of IL-8 and formation of diacylglycerol.

<p>(A) A549 cells were pretreated with various amounts of U73122 or U73343 at 37°C for 60 min, and then incubated with or without alpha-toxin (1.0 μg/mL) at 37°C for 3 h. The concentration of IL-8 in culture supernatants was determined by ELISA. (B) A549 cells were pretreated with various amounts of U73122 or U73343 at 37°C for 60 min, and then incubated with or without alpha-toxin (1.0 μg/mL) at 37°C for 60 min and intracellular DAG levels were determined. Values represent mean ± S.E.; <i>n</i> = 4; *, <i>p</i> < 0.01.</p

Schematic model of alpha-toxin-induced membrane dynamics and accumulation of the GM1a/TrkA complex

<p>Schematic model of alpha-toxin-induced membrane dynamics and accumulation of the GM1a/TrkA complex</p

Transbilayer movement of DAG on the membrane treated with alpha-toxin.

<p>DNA transfection was used to express EYFP-C1AB in A549 cells. After 24 h, cells were incubated with 1.0 μg/mL wild-type alpha-toxin (A) or H148G alpha-toxin (B) at 37°C. The cells were visualized by confocal fluorescence microscopy. Scale bar, 10 μm.</p

Clustering of GM1a and phosphorylation of TrkA in the membrane of cells treated with alpha-toxin.

<p>(A) A549 cells stained with BODIPY-GM1a were incubated with 1.0 μg/mL wild-type alpha-toxin or H148G alpha-toxin at 37°C for 60 min. The cells were fixed in 4% paraformaldehyde and stained with Hoechst 33342. GM1a (green) and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (B) Bodipy-GM1a fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 3; *, <i>p</i> < 0.01. (C) A549 cells were incubated with 1.0 μg/mL wild-type or H148G alpha-toxin at 37°C for 60 min. The cells were fixed, permeabilized, and stained with phospho-TrkA antibody and Hoechst 33342. Phospho-TrkA (red) and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (D) Phospho-TrkA fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 5; *, <i>p</i> < 0.01.</p

Inhibition of endogenous PLC affected the clustering of GM1a and phosphorylation of TrkA.

<p>(A) A549 cells were preincubated with 40 μM U73122 (endogenous PLC inhibitor) or U73343 (U73122 analogue) at 37°C for 60 min. The treated cells were stained with BODIPY-GM1a and incubated with 1.0 μg/mL wild-type or H148G alpha-toxin at 37°C for 60 min. The cells were fixed in 4% paraformaldehyde and stained with Hoechst 33342. GM1a (green) and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (B) A549 cells were preincubated with 40 μM U73122 or U73343 at 37°C for 60 min. The treated cells were incubated with 1.0 μg/mL wild-type or H148G alpha-toxin at 37°C for 60 min. The cells were fixed, permeabilized, and stained with phospho-TrkA antibody and Hoechst 33342. Phospho-TrkA (red) images and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (C) Bodipy-GM1a fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 3; *, <i>p</i> < 0.01. (D) Phospho-TrkA fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 5; *, <i>p</i> < 0.01.</p