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

Fig. 2.

Fig. 2.

Sensitization of cell death by R406 requires the kinase activity of RIPK1. (A) Ripk1−/− MEFs were retrovirally transduced with vectors for empty control and HA-tagged mRIPK1, respectively. The cells stably selected with puromycin were treated as indicated for 15 h and cell death was assessed by CellTiter-Glo assay. Mean ± SEM of n = 3. **P < 0.01; ***P < 0.001; n.s. not significant (Left). The RIPK1 protein levels were analyzed by immunoblotting and equal loading was controlled by determining the tubulin levels (Right). (B) Ripk1−/− MEFs were retrovirally transduced with vectors for empty control, HA-tagged mRIPK1, and the catalytic dead mutants D138N or K45M, respectively. The cells stably selected with puromycin were treated as indicated for 11 h, and cell death was assessed by CellTiter-Glo assay. Mean ± SEM of n = 3. *P < 0.05; n.s. not significant (Left). The RIPK1 protein levels were analyzed by immunoblotting and equal loading was controlled by determining the tubulin levels (Right). (C) Ripk1−/− MEFs were retrovirally reconstituted with an empty vector (EV), HA-tagged mRIPK1, and catalytic dead mutants D138N or K45M, respectively. The cells were subsequently pretreated with or without R406 (3 μM) for 30 min and then stimulated with hTNFα (20 ng/mL) for 2 h. The cells were lysed with RIPA buffer. The cell lysates were analyzed by western blotting with the indicated antibodies. (D) 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 western blotting using antibodies for phosphorylated and total RIPK1, PARP1, Cleaved caspase-8, caspase-8, and Actin as indicated. (E) Ripk1-/- MEFs were retrovirally reconstituted with an empty vector (EV), HA-tagged mRIPK1, and catalytic dead mutants D138N or K45M, respectively. The cells were subsequently pretreated with or without R406 (3 μM) for 30 min, and then hTNFα (20 ng/mL) and zVAD.fmk (20 μM) were added for various time points. The cells were lysed with RIPA buffer. The cell lysates were analyzed by western blotting with the indicated antibodies. (F) 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 and 20 μM zVAD.fmk were added for various time points. The cell lysates were analyzed by western blotting using antibodies for phosphorylated and total RIPK1, RIPK3, MLKL, and Tubulin as indicated. (G) HEK293T cells were transfected with NF-κB firefly luciferase plasmid and Renilla luciferase and subsequently cultured for 20 h. The cells were pretreated with or without 3 μM R406 or 0.5 μM 5Z-7, or 5 μM BAY11-7082 for 30 min and then 10 ng/mL TNFα were added for 60, 90, and 120 min. NF-κB luciferase activity was analyzed by the Dual-Luciferase Assay system.