Ca 074me

Bone marrow-derived macrophages (BMDM) were generated by plating bone marrow in L929 conditioned medium containing M-CSF in 5 cm diameter non-cell culture treated Petri dishes as described previously [18 (link)]. On day 7, BMDM were harvested in ice cold PBS, 5% FBS, 2.5 mM EDTA by incubating 12 min in the fridge and resuspending by pipetting. The cells were then seeded at 1 x 10⁵ cells/well in 96-well plates and rested overnight prior to infection. L. pneumophila used for infection was grown for 3 days at 37°C on CYE agar plates, then inoculated in ACES yeast extract medium at an OD600 of 0.1 and grown for 21 h at 37°C before use, with 5 mg/ml chloramphenicol added to maintain plasmids. BMDM were infected at MOI 0.1, synchronized by centrifugation, and incubated for 3 days at 37°C, 5% CO₂. Intra- and extracellular CFU were quantified on day 3 by plating on CYE plates after a 10 min incubation in dH₂O to lyse BMDM. Where indicated, 20 nM V-ATPase inhibitor bafilomycin A1 (Enzo Life Sciences, BML-CM110-0100), 25 μM cathepsin B inhibitor CA-074-Me (Enzo Life Sciences, BML-PI126-0001), 25 μM cathepsin D inhibitor pepstatin A (Enzo Life Sciences, ALX-260-085-M005), 2 μg/ml TNFR1-Fc (Adipogen, AG-40B-0074-C050), 25 μg/ml anti-IL1β (R&D, AB-401-NA), 25 μg/ml anti-TNF (Bioxcell, BE0058, clone XT3.11) or 100 ng/ml TNF (Peprotech, 315-01A) were added 15 min prior to infection.

ECs were nucleofected with a mCherry-vWF expressing plasmid [5 (link)] by using the Amaxa Nucleofector Technology (Lonza, Milan, Italy). Twenty-four h after nucleofection, cells were serum starved or not for 16 h in endothelial basal medium (EBM) containing 0.5% FBS alone or in combination with 3-MA (5 mM) (Sigma-Aldrich, St. Louis, MO, USA) or a pharmacological inhibitor of cathepsin-β, CA-074Me (50 µM) (Enzo life sciences, Farmingdale, NY, USA). Cells were treated for 1 h with 10 ng/mL of GST, p24 or p17 and fixed with 4% paraformaldehyde. When reported, p17 was preincubated for 30 min at 37 °C with 1 μg/mL of unrelated control mAb (Ctrl mAb) or p17-neutralizing mAb MBS-3. When indicated, serum-starved ECs were treated for 1 h with the PI3K inhibitor LY294002 (10 μM) or WT (100 nM) (Enzo life sciences, Farmingdale, NY, USA) before p17 stimulation. Fluorescence was analyzed using a Leica (Wetzlar, Germany) TCS SP5 laser scanning fluorescence microscope and the imaging software Leica Application Suite. The number of puncta per cell was quantified using ImageJ software, by counting vWF-positive puncta in 20 cells/experiment. Error bars represent the standard deviation calculated as the mean of 3 independent experiments with similar results.

Cathepsin B activity assay was performed as described in the publication by Kubisch et al. [28 (link)]. Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent (78501, Thermo Fisher Scientific) supplemented with protease inhibitors (Complete Mini EDTA-free, Roche), sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride. The fluorogenic cathepsin B substrate Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride (C5429, Sigma-Aldrich) was added to 30 μg of the cell lysate diluted in assay buffer supplemented with 2 mM L-cysteine (C7880, Sigma-Aldrich) and incubated for 30 min at 40°C. Fluorescence was measured at 348 nm (excitation) and 440 nm (emission) with a microplate reader (Varioskan Flash, Thermo Fisher Scientific). The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me (Enzo Life Sciences, Lörrach, Germany) was added to an additional reaction.