Skip to main content
. 2023 Sep 21;120(39):e2308079120. doi: 10.1073/pnas.2308079120

Fig. 1.

Fig. 1.

Identifying small molecule sensitizers of RIPK1-dependent cell death. (A) Chemical structure of R406 and R788. (B) FADD-deficient Jurkat cells were pretreated with or without 3 μM R406 or 3 μM R788 or 10 μM Nec-1s for 30 min as indicated, and then 2 ng/mL TNFα was added for 11 h. The cell viability was measured by CellTiter-Glo assay. Mean ± SEM of n= 3. **P< 0.01; ***P< 0.001; n.s. not significant. (C) FADD-deficient Jurkat cells were pretreated with or without R406 (3 μM) or R788 (3 μM) or Nec-1s (10 μM) for 30 min as indicated, and then 10 ng/mL TNFα was added for various time points. The cell lysates were analyzed by western blotting using antibodies for phosphorylated and total RIPK1, MLKL, and Tubulin as indicated. (D and E) HT-29 (D) and NCI-H1975 cells (E) were pretreated with or without 3 μM R406 or 10 μM. Nec-1s for 30 min as indicated and then 20 ng/mL TNFα with 20 μM zVAD.fmk were added for various time points, separately. The cell lysates were analyzed by phosphorylated and total RIPK1, RIPK3, MLKL, and Tubulin as indicated. (F) NCI-H1299 cells were pretreated with or without 3 μM R406 or 10 μM Nec-1s for 30 min as indicated, and then 20 ng/mL TNFα was added for various time points. The cell lysates were analyzed by phosphorylated and total RIPK1, PARP1 and Tubulin as indicated. (G) Necroptosis was induced in MEFs by the treatment with TNFα (20 ng/mL) and zVAD.fmk (20 μM) together with R406 (3 μM) at indicated times, and cell death was determined by STOX Green. Mean ± SEM of n= 4. ****P< 0.0001. (H) MEFs were pretreated with or without 3 μM R406 or 10 μM Nec-1s for 30 min as indicated, and then 20 ng/mL TNFα with 20 μM zVAD.fmk were added for various time points. The cell lysates were analyzed by phosphorylated and total RIPK1, RIPK3, MLKL, and Tubulin as indicated. (I) RDA was induced in MEFs by the treatment with TNFα (20 ng/mL) together with R406 (3 μM) at indicated times, and cell death was determined by SYTOX Green. Mean ± SEM of n= 4. ****P< 0.0001. (J) MEFs were pretreated with or without 3 μM R406 or 10 μM Nec-1s for 30 min as indicated, and then 20 ng/mL TNFα was added for various time points. The cell lysates were analyzed by phosphorylated-S166 and total RIPK1, Cleaved caspase-3, caspase-3 and Tubulin as indicated.