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. 2021 Jul 20;22(9):e52252. doi: 10.15252/embr.202052252

Figure EV4. RIPK3 is critical for SOCS6‐regulated cell death mechanisms.

Figure EV4

  • A
    Relative expressions of RIPK1 and RIPK3 were analyzed by Western blotting in Socs6 +/+ BMDMs stimulated with Mtb for 0–24 h. Representative blots from n = 3 biological replicates are shown.
  • B
    Socs6+/+ BMDMs were stimulated with Mtb for 0–24 h, and cell lysates were collected and immunoprecipitated using an anti‐RIPK1 antibody. The recruitment of caspase 8, RIPK3, FADD, and MLKL was analyzed by immunoblots. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown.
  • C
    Cell death mechanisms of Mtb‐infected Socs6 −/− BMDMs that were transfected with plasmids expressing Myc‐RIPK3 or Myc‐RIPK3 K51A mutant as indicated. Representative data (from n = 3 biological replicates) are shown as the mean ± SEM of technical replicates.
  • D
    Socs6+/+ BMDMs were transfected with Ripk3 siRNA or Mlkl siRNA for 24 h. Afterward, transfected cells were stimulated with Mtb for 12 h, and cell lysates were collected and immunoprecipitated using an anti‐RIPK1 antibody. The recruitment of caspase 8, RIPK3, FADD, and MLKL was analyzed by immunoblot. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown.
  • E–H
    Cell viabilities (E, data are shown as the mean ± SEM of n = 3 biological replicates), cell death mechanisms [F, data are shown as the mean ± SEM of n = 3 biological replicates. G, representative data (from n = 3 biological replicates) are shown as the mean ± SEM of technical replicates], or Mtb growth rates (H, data are shown as the mean ± SEM of n = 3 biological replicates) of Mtb‐infected Socs6 +/+ BMDMs that were transfected with Ripk3 siRNA or Mlkl siRNA as indicated. Z‐VAD treatment concentration was 20 μM, and the treatment time was 24 h.
  • I
    Cell death mechanisms of Socs6 −/− BMDMs transfected with plasmids expressing Myc‐MLKL and stimulated with Mtb for 36 h. Data are shown as the mean ± SEM of n = 3 biological replicates.
  • J
    Cell viabilities of Socs6 −/− BMDMs transfected with plasmids expressing Myc‐MLKL for 24 h and stimulated with Mtb or z‐VAD for 24 h. Data are shown as the mean ± SEM of n = 3 biological replicates.
  • K
    Mtb growth rates of Socs6 −/− BMDMs transfected with plasmids expressing Myc‐MLKL and stimulated with Mtb for 0–120 h. Data are shown as the mean ± SEM of n = 3 biological replicates.

Data information: ANOVA followed by Bonferroni post hoc test (E, F, H‐K) was used for data analysis. *P < 0.05, **P < 0.01. Abbreviation: n.s., not significant.

Source data are available online for this figure.