Fig 2: LPS caused activation of caspase-3 with concomitant apoptosis and shedding of villus IECs, and relatively spared the crypts. (A) Duodenal sections labeled for active caspase-3 by IHC. Arrowhead indicates a positively labeled apoptotic IEC with unaltered morphology, and arrow indicates a positively labeled IEC with shedding morphology. Scale bars: 200 µm (50×), 25 µm (630×). (B) Example of a duodenal villus 1.5 hours following administration of 10 mg PELPS/kg body weight to a C57BL/6 female mouse. Individual cells were counted along the epithelial monolayer lining one side of a villus (delineated by red line) from base to tip, and therefore referred to as a ‘hemivillus’. Cells were categorized as ‘normal’, ‘apoptotic’ or ‘shedding’. A total of 18–20 hemivilli were analyzed for each intestinal segment for each individual animal. (C) Quantification of apoptotic and shedding IECs in the duodenum, jejunum and ileum (bar graphs), and cell positional quantification of ‘apoptotic’ or ‘shedding’ IECs in the duodenum, jejunum and ileum along villus length (line graphs; 0% villus length represents the villus base, 100% represents the villus tip). (D) Quantification of active-caspase-3-positive cells in villus versus crypt IECs. (E) Villus versus crypt IEC counts. n=6 female mice/group; *P<0.05, comparisons by ANOVA.

Fig 3: Spatial and temporal expression of cleaved caspase-3 in the developing chick auditory brainstem. (A) Summary of cleaved caspase-3 expression (shown in green) from E6 to E13. (B) Expression of cleaved caspase-3 in the brainstem from E6 to E13. Neurofilament (NF) staining (red) shows the developmental progression of axonal tracts, with cleaved caspase-3 expression (green) closely following axonal tract development (merged). At E6 labeling is seen in the peripheral and central projections of the auditory VIIIth nerve fibers. At E7 central projections of auditory nerve fibers and their synaptic target, nucleus magnocellularis (NM), also shows labeled axons. At E9–10 NM axons are labeled. At E11–12 these NM fibers are no longer labeled, but expression is seen in dendrites of their synaptic target, nucleus laminaris (NL). Scale bar for E6 = 100 µm, applies to E6–7. Scale bar for E9 = 100 µm, applies to E9–13.

Fig 4: DISC1-mutant organoids exhibit decreased BRN2 expression and reduced proliferation that is rescued by WNT antagonism.Immunostaining was performed on WT and DISC1-mutant day 19 organoids for EdU incorporation and markers as shown. Expression of cell fate markers TBR2, PAX6, P73, Reelin and polarity markers PKC-?, and acetylated-a-tubulin were grossly unchanged (a–j). However, immunostaining of BRN2 was markedly reduced in DISC1-mutant organoids (k, l). Scale bars: a–d 100 µm, e–h 20 µm, i–j 50 µm, k–l 20 µm. m, n WT and DISC1 exon 8 wt/µ organoids were immunostained at day 19 for markers of apoptosis (TUNEL and Cleaved Caspase 3). o, p Quantification of percentage of DAPI positive nuclei positive for TUNEL or cleaved caspase-3 shows no difference with DISC1 disruption. q–u WT and DISC1-mutant organoids with WNT agonism (CHIR) or WNT antagonism (XAV) were pulsed with EdU for 2 days (culture days 6–7), then fixed, sectioned, and stained for EdU and DAPI at day 19. One representative image for each condition is shown. s Percentage of EdU-positive nuclei were quantified. Data were derived from three independent differentiations. Statistics: o, p Variance was significantly different between conditions, Welch’s t-test. s Variance was significantly different between conditions, one-way ANOVA with Geiser–Greenhouse correction, Sidak’s multiple comparisons test. ***p < 0.001, ****p < 0.0001. Scale bars: m, n, q, p, t, u: 50 µm

Fig 5: Murine-specific apoptosis and necroptosis responses and drug sensitivity in mICOs. (A–C) mICOs were isolated and cultured from mouse livers. mICOs were treated with TNF-a (T, 20 ng/mL) and Smac mimetic (S, 120 µmol/L), with or without Z-VAD-FMK (Z, 50 µmol/L), for 12 hours. (B) Representative bright-field images and fluorescent images with 4',6-diamidino-2-phenylindole (DAPI)/phalloidin staining are shown. Enlarged images from the boxed area are shown in the bottom panels. (C) Staining of active caspase 3 and pMLKL in stimulated mICOs are shown. Enlarged images from the boxed area are shown in the bottom panel. (D and E) Quantification of active caspase 3– and pMLKL-positive cells in fluorescent images was performed using ImageJ (at least 15 high-power fields in each condition). (F) T/S- and T/S/Z-exposed mICOs were co-incubated with Nec-1 (20 µmol/L), Nec-1s (20 µmol/L), GSK872 (20 µmol/L), or NSA (50 µmol/L) for 12 hours. Representative bright-field images and fluorescent images with DAPI/phalloidin staining are shown. (G) mICOs (n = 3) were exposed to T/S or T/S/Z for 12 hours, in the presence or absence of inhibitors at different concentrations. Cell viability was measured using CellTiter-Glo reagent. Relative cell viability was normalized to untreated mICOs (CTR). hICOs (n = 4) were co-incubated with T/S/Z and indicated necroptosis inhibitors for 12 hours. Drug response curves are shown. Data are means ± SD. *P < .05, **P < .01, ***P < .001, by (G, D and E) the Kruskal–Wallis test followed by the Dunn post hoc test. BF, bright field.