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. 2024 Feb 23;43(7):1164–1186. doi: 10.1038/s44318-024-00055-y

Figure 2. Ninj1-deficiency blocks cell lysis and LDH release in ferroptotic BMDMs.

Figure 2

(A–C) LDH release in WT and Ninj1−/− BMDMs treated with 1 mM CuOOH for 5 h (A), 5 μM RSL3 for 6 h (B), or 5 μM ML162 for 5 h (C). (D) Confocal microscopy of WT and Ninj1−/− BMDMs after treatment with nigericin 5μg/mL for 1.5 h, 1 mM CuOOH for 3 h or 5 h, 5 μM RSL3 for 5 h or 5 μM ML162 for 3 h or 5 h. Cells were imaged without fixation to show BMDMs morphology, plasma membrane blebs and balloons (white arrows) and bleb collapse. DRAQ7, a membrane-impermeable DNA-binding dye, was used to track plasma membrane permeabilization. Scale bar: 20 µm. (E) LDH release in WT or Ninj1−/− BMDMs reconstituted with a retroviral vector expressing WT or mutant NINJ1 and treated with 1 mM CuOOH for 2 h. Reconstitution with a GFP-expressing vector was used as a control. (F) LDH release in WT or Ninj1−/− BMDMs treated with 1 mM CuOOH for 2 h. (G, H) Cell viability (G), measured based on the absorbance of intracellular ATP signal, and LDH release (H), in WT and Ninj1−/− BMDMs upon treatment with 1 mM CuOOH for 1, 2, 3, 4, or 5 h. When indicated, 25 μM Fer-1 was added simultaneously with ferroptosis and pyroptosis activators (A–D). Data information: All graphs show the mean ± SD. Data are representative of two (D) or three (F) independent experiments (D) or pooled from three independent experiments performed in triplicate (A–C, E, G, H). Statistical analysis was done using two-way ANOVA (A–C) or one-way ANOVA with multiple comparisons tests (E, G, H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.