Vectashield solution containing 4 6 diamidino 2 phenylindole dapi

The cells were plated on sterile coverslips in 6-well plates and treated with 10 µM 2-ClHDyA in 2% FBS for 30 min. The cells were washed quickly with PBS and then were fixed with formalin for 10 min. The cells were permeabilized with 0.25% Triton X-100 for 10 min. The cells were washed with 2% (w/v) BSA in PBS. They were then labeled with 5 μM azide–carboxytetramethylrhodamine (azide–TAMRA) (Sigma-Aldrich; catalog no. 760757) by using the Click-It Cell Reaction Buffer Kit (Thermo Fisher, Waltham, MA, USA; catalog no. C10269) following the manufacturer’s protocols. The click reagents were washed away with 2% BSA in PBS. The cells were then incubated with primary antibodies against COXIV (1:500), GM130 (1:142) and calnexin (1:1000) overnight at 4 °C. The next day, the cells were washed three times with PBS for 5 min to remove any unbound primary antibody. The cells were incubated with the goat anti-mouse IgG secondary antibody (1:500) labeled with Alexa 488 or the goat anti-rabbit IgG secondary antibody (1:500) for 1 h. The coverslips were mounted onto microscope slides with a Vectashield solution containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Newark, CA, USA; catalog no. H1200).

For staining of bacteria and actin, HMEECs were cultured in 8-well chamber slides and infected with P. aeruginosa for varying time periods. After incubation, cells were washed three times with PBS buffer and then fixed and permeabilized with BD cytofix and cytoperm reagent (BD Biosciences, San Jose, CA, USA) for 30 min. After washing, the cells were blocked with 3% normal goat serum (NGS) for 30 min and then incubated with anti-Phospho PKC-α antibody (Abcam, Cambridge, MA, USA) for 45 min followed by Alexa Fluor 488 antibody (Life Technologies, Carlsbad, CA, USA). After washing, cells were counterstained for actin with rhodamine phalloidin (Life Technologies, Carlsbad, CA, USA) for 45 min, washed and mounted in an antifade Vectashield solution containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). The cells were viewed with a Zeiss LSM 710 microscope (Carl Zeiss, Germany) and images were assembled using Adobe Photoshop 7.0.

For staining of bacteria and actin, HMEECs were cultured in 8-well chamber slides and infected with P. aeruginosa for varying time periods. After incubation, cells were washed three times with PBS buffer and then fixed and permeabilized with BD cytofix and cytoperm reagent (BD Biosciences, San Jose, CA) for 30 min. After washing, the cells were blocked with 3% normal goat serum (NGS) for 20 min and then incubated with anti-Pseudomonas aeruginosa antibody (Abcam, Cambridge, MA) for 45 min followed by Alexa Fluor 488 antibody (Life Technologies, Carlsbad, CA). After washing, cells were counterstained for actin with rhodamine phalloidin (Life Technologies, Carlsbad, CA) for 45 min, washed and mounted in an antifade Vectashield solution containing 4, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). The cells were viewed with a Zeiss LSM 710 microscope (Carl Zeiss, Germany) and images were assembled using Adobe photoshop 7.0.