Htb 54

Human embryonic kidney 293T (Clontech; 632180), human lung carcinoma A549 (ATCC; CCL-185), and human hepatoma Huh7.5 (Apath) cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen; 10-013-CV); African green monkey kidney Vero (ATCC; CCL-81) and human lung epidermoid carcinoma Calu-1 (ATCC; HTB-54) cells were cultured in Eagle’s minimum essential medium (ATCC; 30-2003) and McCoy’s 5a medium Modified (ATCC; 30-2007), respectively. All of the media were supplemented with 10% fetal bovine serum (FBS) (Invitrogen; 10082147) and 1% penicillin/streptomycin. Cells were grown at 37 °C with 5% CO₂.

To characterize the Alt a 1 traffic, non-polarized Calu-3 cells (ATCC; HTB-54; USA) were grown on coverslips until reached a 70% of confluency. Then, they were incubated with Alt a 1 at 37°C, 5%CO2 for different times: 5 min for early endosome assay, and 30 min for recycling endosome assays. After incubation, cells were washed, fixed with 4% PFA for 10 min and permeabilized with PBS-Triton X100. Cells were blocked and then they were incubated with anti-Alt a 1 antibody and the proper anti-endosome marker (all antibodies listed in Supplementary Table 1) for 1h at RT. After using Alexa-conjugated secondary antibodies, cells were stained with DAPI and mounted with ProLong Gold over slides. Images were obtained with a Zeiss LSM 880 confocal microscope, using 405, 488 and 633 laser excitations and 63X amplification.
The recycling pathway was inhibited by adding 100 mM endosidin-2 (ES-2; Sigma-Aldrich) 1h prior to adding Alt a 1 to Calu-3 monolayers. Alt a 1 accumulation was evaluated by immunofluorescence following the earlier described protocol.