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. 2020 Mar 16;130(4):2111–2128. doi: 10.1172/JCI133264

Figure 7. mTOR regulates microbial PAMP– and TNF-induced necroptosis.

Figure 7

(A) Immunoblots of protein lysates of ex vivo organoids treated with 10 ng/mL rapamycin for 48 hours. (B and C) Organoid viability was quantified by cellular ATP levels after treatment with 10 ng/mL mTNF or 10 μg/mL poly(I:C) alone (B), or in combinations with 10 nM BV-6 (S)/20 μM z-VAD (Z) or 20 μM z-VAD, respectively (C), for 24 hours (n = 4). (D) WT and Tsc1–/– MEFs were treated with 10 ng/mL mTNF (T), 100 nM BV-6 (S), and 20 μM z-VAD (Z) with or without 10 μM Nec-1 for 6 hours; or treated with 50 μg/mL poly(I:C) (P) and 20 μM z-VAD (Z) with or without 5 μM GSK′872 for 16–20 hours. Cell cytotoxicity was measured by LDH release (n = 3). (E–G) MEFs were treated with 50 μg/mL poly(I:C) (P) and 20 μM z-VAD (Z) (E) or 10 ng/mL mTNF (T), 100 nM BV-6 (S), and 20 μM z-VAD (Z) (F) or 10 ng/mL mTNF alone (G) for various times as indicated. p-RIPK1 (S166), p-RIPK3 (S232), p-MLKL (S345), p-S6 (S235/236), p-IκBα (S32), p-IKKα/β (S176/180), p-p38 (Thr180/Tyr182), and p-Jnk (Thr183/Tyr185) were used. Numbers under Western blot bands represent relative quantifications over actin. Data are representative of 3 independent experiments and are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by unpaired Student’s t test.