Figure 7: Cellular RNA leaks from irradiated crypt cells in a p53-dependent manner and induces TLR3-mediated crypt cell death.

(a) HEK293 cells transiently transfected with a human TLR3 expression vector together with ISRE-luciferase reporter plasmid (HEK293-hTLR3) were incubated with homogenate from irradiated small intestine or homogenate treated with pronase, DNase or RNase. Reporter activity was measured in triplicate. Results are means±s.e.m. *P<0.05 (unpaired two-tailed Student’s t-test). (b) Quantification and size distribution of total RNA in the culture supernatants of crypt–villus organoids of p53^+/+ and p53^−/− mice on day 1 after irradiation. (c) Representative images of crypt–villus organoids of Tlr3^+/+ and Tlr3^−/− mice on day 1 after irradiation. Crypts were treated without or with RNase. Scale bar, 200 μm. The right-hand panel shows the viability. Viability was measured in triplicate. Results are means±s.e.m. N.S., not significant. *P<0.05 (unpaired two-tailed Student’s t-test). (d) HEK293-hTLR3 cells were incubated with medium alone (Med), purified RNA or poly I:C for 12 h. Reporter activity was measured in triplicate. Results are means±s.e.m. *P<0.05; **P<0.01 (unpaired two-tailed Student’s t-test). (e) Representative images of in vitro cultured crypts of Tlr3^+/+ and Tlr3^−/− mice (left) and the viability (right) on day 2 after treatment with Med or purified RNA. Scale bar, 200 μm. Viability was measured in triplicate. Results are means±s.e.m. *P<0.05; **P<0.01 (unpaired two-tailed Student’s t-test). Results are representative of three independent experiments.