Extended Data Figure 9: TNF-independent death of organoids and no direct cell death induction by TLR4 or TLR2 ligand.

Intestinal organoids were derived from small intestine crypt cells isolated from wild-type (WT) and Ripk1^iIEC-KO mice. RIPK1 deletion (iKO) was induced in vitro by 4-OHT treatment (200 nM) for 20−24 h. a, Organoids were observed for up to 4 days after removal of 4-OHT. b, Organoids were treated with 10 ng ml⁻¹ mTNF, Gram-negative bacterial lipopolysaccharide (TLR4 ligand, 100 µg ml⁻¹), or Gram-positive bacterial lipoteichoic acid (TLR2 ligand, 100 µg ml⁻¹) after removal of 4-OHT. Time-lapse confocal microscopic imaging of propidium iodide (PI)-stained organoids (n = 2). Scale bar, 100 µm. c, Image analysis of the percentage of propidium-iodide-positive nuclei in an experiment resembling those in b.