Figure 1. Ferroptosis activators induce NINJ1 oligomerization.

(A–C) Lactate dehydrogenase (LDH) release in WT 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–F) Histograms of BODIPY 581/591 C11 fluorescence at 540 nm emission wavelength, oxidized form (BODIPY-C11^Ox), showing cell counts in WT BMDMs left untreated (UT) or after 30 min of treatment with 1 mM CuOOH (D), 5 μM RSL3 (E), or 5 μM ML162 (F). (G) Immunofluorescence confocal microscopy of endogenous NINJ1 (green) in PFA-fixed WT BMDMs after treatment with 5μg/mL nigericin for 1.5 h, 1 mM CuOOH for 3 h, 5 μM RSL3 for 5 h or 5 μM ML162 for 5 h. White squares indicate inset images, and the arrows highlight NINJ1 oligomers. Scale bar: 10 µm. (H–J) Western blot analysis of endogenous NINJ1 in BMDMs treated with 1 mM CuOOH for 3 h (H) 5 μM RSL3 for 5 h (I) or 5 μM ML162 for 4 h (J) in the presence or absence of 25 μM Fer-1 followed by treatment with the membrane-impermeable crosslinker BS³(+). Mixed supernatant and cell extracts were analyzed. Tubulin (TUB) serves as loading control. FL, full length; Short exp = short exposure; 1-mer = monomer; 2-mer = dimer; 3-mer = trimer and n-mer: higher-order oligomers. (K, L) Fluorescence confocal microscopy of HeLa cells expressing hNINJ1-GFP (K) or HA (Hemagglutinin)^TMD-GFP (L) treated with CuOOH for 3 h. Images show green fluorescence at the basal or central plane of the cell. DRAQ7 (maximum projection from a Z-stack), a membrane-impermeable DNA-binding dye, was used to track plasma membrane permeabilization. Scale bar: 20 µm. When indicated, 25 μM Fer-1 was added simultaneously with ferroptosis or pyroptosis activators (A–J). Data information: Graphs show the mean ± SD. Histograms show cell counts normalized to mode. Data are representative of three (A–F), two (H–J) or at least five (G, K, L) independent experiments performed in triplicate. Source data are available online for this figure.