Cytochalasin D (Zygosporin A) inhibits G-actin–cofilin interactions by binding to G-actin. Cytochalasin D also inhibits cofilin binding to F-actin and decreases actin polymerization and depolymerization rates in live cells. In the actin polymerization inhibit assay, A549 cells treated with cytochalasin D at a working concentration of 500 nM and then labeled F-Actin and the nuclei with the live cell fluorogenic F-actin-labeling probes SiR-actin (CY-SC001; Cytoskeleton, Inc.; red) and Hoechst 33342 (blue), respectively.
To investigate the functions of F-actin on adhesion and invasion of M. catarrhalis, A549 cells were incubated with different concentrations of cytochalasin D (100 nM, 500 nM, 1 μM, 5 μM, and 10 μM) in 0.2% DMSO for 45 min before infection with M. catarrhalis 73-OR isolate. Control wells contained only 0.2% DMSO. Gentamicin (300 μg/mL) was added to the infected monolayer cells before lysing with 0.25% trypsin and 0.1% saponin to kill any remaining adherent extracellular bacteria. The samples were plated on blood agar plates containing 5% sheep blood to determine the number of adherent or invasion bacteria. All assays were performed in six replicates. At least three independent experiments were performed.
To investigate the functions of F-actin on adhesion and invasion of M. catarrhalis, A549 cells were incubated with different concentrations of cytochalasin D (100 nM, 500 nM, 1 μM, 5 μM, and 10 μM) in 0.2% DMSO for 45 min before infection with M. catarrhalis 73-OR isolate. Control wells contained only 0.2% DMSO. Gentamicin (300 μg/mL) was added to the infected monolayer cells before lysing with 0.25% trypsin and 0.1% saponin to kill any remaining adherent extracellular bacteria. The samples were plated on blood agar plates containing 5% sheep blood to determine the number of adherent or invasion bacteria. All assays were performed in six replicates. At least three independent experiments were performed.