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. 2022 Jan 12;5:47. doi: 10.1038/s42003-021-02953-x

Fig. 1. Mouse Serpinb3a protected fetal intestinal epithelial cells (FIECs) from lysoptosis-like death.

Fig. 1

a, b mSerpinb3a+/+ (blue bars with individual data points marked) or mSerpinb3a−/− (red bars with individual data points marked) FIECs were incubated in 100% (a) or 25% (b) DPBS for the indicated time points. The percent dead (Sytox positive) was calculated as (# Sytox positive nuclei/# of DAPI positive nuclei) × 100. The mean ± SD were from counts covering at least seven fields and compared using a two-tailed t-test (***P < 0.001). A representative experiment is shown. c–l Live-cell, confocal microscopy assessed lysosomal content (red, 10 K TMR-labeled dextran), and plasma membrane permeability (green, SG) in mSerpinb3a+/+ (c–g) or mSerpinb3a−/− (h–l). FIECs incubated in 25% (c–f, h–k) or 100% DPBS (g and l). Z series were obtained every 2 min for 1 h, indicated time points are shown (scale bar = 25 µm); inset scale bar = 5 µm). Note marked loss of endolysosomal staining in SG positive mSerpinb3a−/− versus mSerpinb3a+/+ FIECs (k vs. f). FIECs incubated in 100% DPBS did not show SG uptake or loss of TMR-labeled dextran (g, l). Representative transmission electron micrographs (TEM) of mSerpinb3a+/+ (m–p) or mSerpinb3a−/− (q–t) FIECs after incubation in 25% DPBS (m, n, o and q, r, s) or 100% DPBS (p and t). Low magnification (scale bar =2 µm) TEM images of mSerpinb3a+/+ or mSerpinb3a−/− FIECs (m, p and q, t, respectively) show cell morphology. High magnification (scale bar =100 nm) TEM images (n, o and r, s, respectively) demonstrate cell morphology. n and r were magnified cells from within the hashed box in m and q, respectively. o and s were individual cells from a different field of view. Some mSerpinb3a+/+ FIECs exhibit hallmarks of apoptotic morphology (n), including chromatin condensation (black arrowheads) and plasma membrane budding (open arrowheads), or necrotic morphology (o), including plasma membrane breaks (black arrows) and organelle degeneration (open arrows). mSerpinb3a−/− FIECs displayed a necrotic cell death morphology (black and open arrows as above) along with severe vacuolization and cytoplasmic clearing (asterisks) (r, s). Original imaging data files can be found at https://data.mendeley.com/datasets/hk2t9x7d6x/1.