Fig. 6: Lysosomal hyperactivation and LMP in ECs treated with HLA-DR antibody.

a–d After pretreatment with IFN-γ for 4 days, HUVECs were incubated with the indicated antibodies for 3 h. Cells were then stained with (a) Lysotracker Red to measure lysosomal appendency, (b) Lysosensor Green to measure lysosomal acidity, and (c, d) Magic Red for cathepsins B and L activity, respectively (N = 3). a–d MFI of stains was measured by flow cytometry. Data are presented as mean ± SEM. *P < 0.05. e HUVECs were treated with L243 for 3 h and then stained with Magic Red to determine cathepsin L activity. A representative image of three independent experiments is shown (Bars = 20 µm). f After pretreatment with IFN-γ for 4 days, HUVECs were loaded with AO (10 µg/ml), after which cells were treated with L243 and microscopic images were taken. Data show a representative image from three independent experiments. The line chart shows the AO red fluorescence intensity from time-lapse recording for up to 100 min. g HUVECs were treated with L243 for 3 h and stained with Lysotracker Red and annexin V FITC (N = 3). Cells were subjected to flow cytometry to determine the relationship between lysosomal volume and cell death. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01. h–j After pretreatment with CA-074 for 30 min in order to block cathepsin B activity, HUVECs were incubated with L243 for 3 h (N = 4). h, i For evaluation of mitochondrial alterations, adherent cells were loaded with Mitotracker and MitoSOX before analysis. j Cytotoxicity was assessed by flow cytometry with PI (■) and annexin V (□). **P < 0.01 significant differences CA-074 plus L243 versus L243 alone for both ■ and □. IsoCtrl isotype control, MFI mean fluorescence intensity, NT non-treated, CA-074 CA-074 Me