Fig. 7: Initiation of mTP-dependent necrosis by H2O2 triggered lysoptosis-like death in HT3B3-KO cells.
From: Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins
a–n Representative live-cell confocal images of HT3B3-WT and HT3B3-KO cells stained with JC-1, then treated with 5 mM H2O2. Images were collected using Ex 488 nm with detection windows of Em 498–532 nm (JC-1 monomer, green) and 566–606 nm (JC-1 oligomer, red) every 5 min over 3 h (select time points shown). m Fluorescence intensity of JC-1 oligomer/JC-1 monomer normalized to time 0 (rel. JC-1 oligomer/JC-1 monomer) of the experiment in panels a–n. Time points with no fluorescence in either channel (dead cells) were eliminated from the analysis. n Area under the curve (AUC) for multiple fields for experiment in panels a–n (compared using one-way ANOVA with Tukey multiple comparisons). o Percent dead ((#Sytox™ positive/# of nuclei) × 100) of HT3B3-WT or HT3B3-KO cells treated with 5 mM H2O2 or DPBS for 4 h. p Percent dead HT3B3-WT or HT3B3-KO cells pretreated with DMSO, 10 µm cyclosporin A (CsA), or E64d for 1 h prior to exposure to 5 mM H2O2 or DPBS for 4 h. q–t To quantify lysosomal content, HT3B3-WT or HT3B3-KO cells were incubated with fluorescently labeled different molecular weight dextrans as above and imaged using live-cell confocal microscopy. The number of lysosomes at each time point (LCt) were normalized to the lysosome count at time zero (LCt0). u AUC for each dextran over time for an experiment in q–t (compared by two-way ANOVA with Tukey’s multiple comparisons test). v–y Representative TEM images (scale bars = 2 µm) of HT3B3-WT or HT3B3-KO cells treated with DPBS or 5 mM H2O2 for 4 h. Insets show normal or abnormal mitochondria (open and dark arrowheads, respectively; scale bars = 500 nm). Yellow arrowheads indicate enlarged vesicle and cytoplasmic clearing. (z-aa) Quantification of TEM images from the experiment in v–y showing the number of necrotic cells per field (z) and abnormal mitochondria per field (aa) (≥5 fields). Unless otherwise noted, a representative of ≥3 replicates is shown, and the means ± SD were compared using a two-tailed t-test (ns not significant, ***P < 0.001, **P < 0.01, *P < 0.05). Original data files can be found at https://data.mendeley.com/datasets/tpp7xnmwxg/1.
