Fig. 2. LMP and lysosomal cysteine peptidases, but not executioner caspases or RIPK1, induced lysoptosis-like death in mSerpinb3a^−/− FIECs.

a-x Representative confocal fluorescence images of mSerpinb3a^+/+ (a-l) or mSerpinb3a^−/− (m-x) FIECs pretreated with diluent (DMSO; a-d; m-p), 2 µm DEVD-CHO (e-h; q-t) or 2 µm E64d (i-l; u-x) and then incubated in 25% DPBS for 1 h (scale bar = 40 µm). FIECs were stained with Hoescht 33342 (H33342; blue), annexin V-FITC (annexin V, green), propidium iodide (PI, red), and Alexafluor⁶⁴⁷ conjugated dextran (dextran⁶⁴⁷, white). y, z Quantification of dead cells (PI-positive; y) or lysosomal area (dextran⁶⁴⁷ staining; z) from different representative experiments (n = 3) over multiple fields (n > 5). i-xii Confocal maximum intensity projections of fluorescently labeled lysosomes from mSerpinb3a^+/+ (i–vi) or mSerpinb3a^−/− (vii–xii) FIECs (scale bar = 25 µm). Cultures were pretreated with DMSO (i-iii; vii-ix) or nec-1 (iv-vi; x-xii) for 1 h prior to incubation in 25% DPBS. Cell numbers, cell death, and lysosomes were quantitated using H33342 (blue), PI (red), and dextran⁶⁴⁷ (white), respectively. Representative images are from the same experiment, which was repeated three times. xii, xiv Quantification of PI positive (xii) or lysosomal (xiv) staining from multiple fields (n ≥ 6). The means ± SD of a representative experiment were compared using a two-way ANOVA with Tukey’s multiple comparisons (**P < 0.01, ***P < 0.001). Original data files can be found at https://data.mendeley.com/datasets/scgbb3s333/1.