Lysotracker dye

For analyzing viability, cells were stained with an annexin-V and propidium iodide (Thermo Fisher, 88-8007-74) apoptosis detection kit according to the manufacturer’s instructions. For analyzing lung infiltration, lungs were intubated ex vivo through the trachea with 2 ml dispase solution (5 U ml⁻¹) and then agitated at 37 °C. Lungs were chopped and dissociated in 1% BSA + 60 U ml⁻¹ DNase1 + 70 U ml⁻¹ collagenase type 1 dissolved in PBS with the help of a GentleMACS system (Miltenyi) according to the manufacturer’s instructions. RBCs were removed in ammonium-chloride-potassium lysis buffer and debris was removed using cell strainers. Cells were stained on 4 °C in PBS with 1% FBS using a 1:300 dilution of anti-CD45-PE, anti-CD45-APC, anti-CD11b-PE/Cy7 (all from BD) and anti-Ly6G-PE, anti-CD11b-Pacific blue and F4/80-APC (all from BioLegend) in the presence of a blocking antibody, rat anti-mouse CD16/CD32 (BD; 1:600 dilution). Labile iron was measured using the FerroOrange Dye (F374, Dojindo), total ROS was measured using a total ROS detection kit (ENZ-51011, Enzo) and lysosomal mass was assessed by using a LysoTracker dye (L12492, Invitrogen), all according to the manufacturer’s instructions. Samples were acquired on a Gallios flow-cytometer (Beckman Coulter) and acquired data were analyzed with FlowJo software (Tree Star).

Cells (treated or untreated) were incubated with Mitotracker dye (75 nM, Invitrogen), Lysotracker dye (100 nM, Invitrogen) and Hoechst (10 µg/ml) in complete medium and incubated for 30 min at 37 °C in 6 well plate. The cells were then washed with 1X PBS for three times and phenol red free complete media was added for imaging under live cell condition. Imaging was done using florescent microscopy (Zeiss Microsystems, GmBH, Germany).

Caco-2 cells (2 × 10⁶ cells) were seeded in chamber slides (Nunc, Lab-tek, ThermoFisher) and after 15 days of culture incubated with recombinant purified U-Omp19 at different concentrations (50 or 100 μg/ml) or Leupeptin (10 μg/ml) for 3 h at 37 °C, 5% CO₂. Then, 50 μM of cathepsin L specific fluorogenic substrate (CBZ-Phe-Arg)₂ and 50 nM Lysotracker dye (Invitrogen) were added in HBSS medium for 30 min at 37 °C, 5% CO₂. Proteolytic activity of cathepsin L was evaluated by confocal microscopy in living cells. Each chamber was evaluated in at least five random fields, the microscope used was an IX-81 attached with a FV-1000 confocal module (Olympus Corp.) and an Objective 60× PlanApo (Olympus Corp.). Colocalization analysis was made with ImageJ (NIH, US) and ICY Image Software (Institute Pasteur, France). ImageJ Plugging using a Rolling Ball of 50 pixels was used to subtract background of each image. Measurements of the maximum and minimum value of Threshold for each image were performed using ImageJ. The ICY Image Software was used to set the ROIs using the Maximum and Minimum values and then Manders coefficient was calculated for each image.

The Cyto-ID (ENZO LIFE SCIENCES ENZ-51031-K200, Farmingdale, NY, USA) is an 488 nm-excitable green fluorescent reagent that specifically accumulates in autophagic vesicles. Cells were incubated in Cyto-ID (1 μl Cyto-ID/1ml cell culture medium) for 30 minutes at 37 ^oC, 5% CO₂ and washed prior to analysis. Lysotracker dye (DND-99 LIFE TECHNOLOGIES, Oregon, USA) was incubated for 20 min at 37ºC. Cells were analyzed by Fluorescence Nikon ECLIPSE Ti-U microscope.

MDA-MB-231 cells and SKBR3 cells are plated in a 96 wells plate at 7500 cells per well. After 36 h, cells were washed with 100 µl of Fluorobright DMEM (Life technologies, France) to remove the phenol red. The lyso-tracker dye (Life technologies, France) was diluted 10,000 times, the cell mask dye was diluted 1000 times and were incubated together 30 min at 37 °C. The nucleus staining was performed with 0.5 µg/ml of Hoechst 3342 (Life technologies, France) and then incubated 10 min at 37 °C. The plate was washed again with 100 µl of fluorobrite DMEM. The antibody was injected and the kinetic was immediately recorded at 37 °C by an HCS Operetta microscope (Perkin Elmer) during 3 h. Control were performed on ice in order to block the cell metabolism. After 3 h the plate was washed with PBS and the cells were fixed with 150 µL of paraformaldehyde at 4 °C for 15 min. The plate was washed with 150 µl of PBS and imaged by confocal microscope (LSM 780 Confocal microscope, Zeiss, France).

Cells were cultured on cover slips and exposed to GD/GR. After treatment Lysotracker dye (Life technologies, DND-99) was incubated for 20 min at 37 °C. Confocal images (Leica TCS SP5 using 63 × water immersion objective with UV-405 nm laser for Hoechst and He/Ne-543 for Lysotracker) were taken after 20 h of GD. Hoechst was used as a nuclear counterstain. The count of Lysotracker-positive vesicles was performed by FIJI image analysis software,^{41 (link)} considering three independent experiments with three repeated technical samples. We analyzed the 10 last images of the total stack to obtain the maximum projection and gauged the parameters of ‘analyze particles’ plug-in as follows: size (area) from 0.2 to 25 μm² and circularity from 0.1 to 1.0 for identification of positive vesicles.