Triton x

HeLa cells (LKB1-null) were co-transfected with either pEGFP-C1 control or our panel of LKB1 constructs and Flag-STRADα using Lipofectamine 2000 (Invitrogen), per manufacturer’s protocol. 24 hours later, cells were fixed using Phemo buffer with a final concentration of: 3.7% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), 0.05% glutaraldehyde (MP Biomedicals, Santa Ana, CA), and 0.5% Triton-X (Promega, Madison, WI) for 10 minutes at room temperature. Fixed cells were rinsed with PBS and stained with Alexa Fluor^® 555 Phalloidin (1:200 in PBS) for 1 hour, rinsed three times with PBS, and stained with 350 nM DAPI for 10 minutes followed by three more PBS washes. Coverslips were then mounted with ProLong^® Diamond Antifade Mountant. Fixed cells were imaged using a Leica SP8 inverted confocal microscope at 63x (HP PL APO 1.40 NA oil) using a 488 and 514 nm argon laser.

Endothelial cells were dissociated to single cells for immunocytochemistry analysis through the use of TrypLE Express. Cells were then fixed using 4% PFA (Nacalai-Tesque, Japan) for 15 min at room temperature, followed by 0.2% Triton-X (Promega, U.S.A.) treatment for 15 min to permeabilise the cells when required. The cells were then treated with blocking buffer (PBS with 2% BSA and 5% Fetal Bovine Serum; Gibco, U.S.A.) for 30 min. Primary antibodies were diluted accordingly and incubated at 4 °C overnight. Cells were washed and then incubated with secondary antibodies conjugated with the appropriate Alexa Fluor® fluorescent dyes for 1.5 hours at 4 °C. Cells were then counter-stained with 4,6-diamindino-2-phenylindole (DAPI) (AAT Biorequest, U.S.A.) for nuclei visualisation. For this study, primary antibodies used are mouse anti-human CD31 (1:200), rabbit anti-human VCAM-1 (1:200), rabbit anti-human vWF (1:200), mouse anti-human eNOS (1:200), mouse anti-ATP5B (1:500), mouse anti-TOMM20 (1:500), rabbit anti-human Ki67 (1:1000), rabbit anti-human VeCAD (1:200) and mouse anti-human MTCO2 (1:200). Secondary antibodies conjugated with Alexa Fluor® 488 or 594 were then used for fluorescence detection. Cells were viewed and imaged using the Olympus Fluoview inverted confocal microscope (Olympus, U.S.A.).