Extended Data Fig. 9: RIPK1 S321 phosphorylation is important for inhibiting LPS/5z7-induced pyroptosis.

a, Confirmation of the expression of WT RIPK1 and different mutants in Ripk1-KO BMDMs by immunoblotting. b, RIPK1-WT or mutant-reconstituted BMDMs were treated with LPS (50 ng/ml) and 5z7 (200 nM) for the indicated time. Cell death was measured by SytoxGreen positivity assay. c, Primary Ripk1^WT/WT and Ripk1^S321E/S321E BMDMs were challenged with YopJ-deficient Y. pseudotuberculosis (MOI 40) for 4 hours. Cell death was measured by LDH release. d, Primary Ripk1^WT/WT and Ripk1^S321E/S321E BMDMs were challenged with Y. pseudotuberculosis (MOI 40) for 1 hour. The amount of bacteria taken up by cells was quantified by CFUs. e, Primary Ripk1^WT/WT and Ripk1^S415A/S415A BMDMs were treated with LPS (50 ng/ml) and 5z7 (200 nM) for the indicated time. Cell death was measured by SytoxGreen positivity assay. f, Primary Ripk1^WT/WT and Ripk1^S415A/S415A BMDMs were treated with LPS (50 ng/ml) and zVAD (50 μM) for the indicated time. Cell death was measured by SytoxGreen positivity assay. g, Eight-week-old male Ripk1^WT/WT and Ripk1^S321E/S321E mice were subjected to intragastric gavage with Y. pseudotuberculosis (1 × 10⁷ CFU). The survival ratio of mice was monitored at desired stages. n = 5 mice for each group. Two-sided log-rank (Mantel-Cox) test. Mean ± s.d. of n = 3 (b, d and f), 5 (c) or 4 (e) independent wells of one representative experiment.

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