Figure 2.

Distinct features of necroptotic cell death in hICOs. (A) Apoptosis and necroptosis were induced in hICOs, PSC-ICOs, and ALD-ICOs using different combinations of TNF-α (T, 20 ng/mL) and Smac mimetic (S, 120 μmol/L), with or without Z-VAD-FMK (Z, 50 μmol/L). (B) Cell viability measurement was performed using CellTiter-Glo reagent. Relative cell viability was calculated by normalizing to dimethyl sulfoxide–treated hICOs (CTR) after 6 hours of incubation (n = 5). (C) The diameter of stimulated hICOs was determined in a time-course manner and analyzed using ImageJ software. (D) Real-time, live-cell imaging of stimulated hICOs incubated with 100 ug/mL PI (red). (E) Representative bright-field images of stimulated hICOs, ALD-ICOs, and PSC-ICOs after 6 hours. (F and G) Time-course cell viability of stimulated hICOs (n = 6), ALD-ICOs (n = 3), and PSC-ICOs (n = 3). (H) Confocal imaging of the hICOs treated for 6 hours after staining with 4′,6-diamidino-2-phenylindole (DAPI)/phalloidin. Enlarged images from the boxed area are shown in the bottom panel. (I) Representative TEM images of stimulated hICOs at a time point of 6 and 12 hours. Typical cell death–related characteristics were found as indicated by arrows (pink, pyknosis; dark green, shrunken cytoplasm; purple, fragmented nucleus; yellow, plasma membrane blebbing; light green, cytoplasmic vacuolization; red, condensed mitochondria; dark blue, karyolysis; light blue, ruptured plasm membrane; white, rounded nuclei). Data are means ± SD. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 by (C, F, G) Mann–Whitney test and (B) the Kruskal–Wallis test followed by the Dunn post hoc test.