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. 2023 Nov 18;14(11):755. doi: 10.1038/s41419-023-06284-z

Fig. 5. Reactive oxygen species are neither sufficient nor required to induce NINJ1-dependent plasma membrane permeabilization.

Fig. 5

A, B V5-NINJ1 reconstituted Ninj1 KO MEFs were treated with doxycycline overnight and the next day stimulated ML162 for the indicated duration (A, B) in the presence or absence of 30 min pretreatment with Fer-1 (B). NINJ1 oligomerization was determined by immunoblotting after BS³ crosslinking. The results are representative of at least two independent experiments. C–J Wild-type and Ninj1 KO MEFs were pretreated for 30 min with DMSO, Fer-1 or Trolox, and then stimulated with the indicated compounds. PMP was determined by Sytox Green positivity in function of time (C, D, F, G, I, J) or by LDH release at the indicated timepoint (E, H). K, L Wild-type (K, L) or Ninj1 KO MEFs (L) expressing an inducible shRNA construct against GPX4 were treated for 48 h with doxycycline in the presence of Fer-1. After 48 h, Fer-1 was either reapplied (K) or removed (K, L) and PMP was determined by Sytox Green positivity in function of time. M, N Wild-type or Gsdme KO MEFs were pretreated for 30 min the presence or absence of Trolox and then stimulated with MNNG (M) or etoposide (N). PMP was determined by Sytox Green positivity in function of time. Data in the graphs are presented as mean ± SEM of independent experiments (n = 3). Statistical significance was determined unpaired two-tailed T tests (E, F, H, I), one-way ANOVA with Dunnett post hoc testing (C) or by two-way ANOVA (D, G, J–M, P). **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.