<p>Recombinant Sph protein induces ferroptotic cell death, which is counteracted by ferroptosis inhibitors.</p>

<p><b>(A)</b> LDH release from MAC-T cells treated with the indicated concentrations of recombinant Sph for 6, 12, or 24 h under continuous incubation at 37°C (n = 5). <b>(B)</b> LDH release from MAC-T cells treated with recombinant Sph under the same conditions, followed by an additional 1-h incubation at 4°C (n = 5). <b>(C)</b> Representative immunoblots showing SLC7A11 and GPX4 expression in MAC-T cells treated with recombinant Sph (4 or 32 μg/mL) for 6 h, with corresponding quantitative analyses (n = 3). <b>(D)</b> Schematic diagram of the experimental protocol for ferroptosis inhibitor intervention in recombinant Sph-treated MAC-T cells.

(E-I) LDH release from MAC-T cells treated with recombinant Sph (4, 16, or 32 μg/mL) in the presence of the indicated concentrations (0-30 μM) of ferrostatin-1 <b>(E)</b>, zileuton <b>(F)</b>, troglitazone <b>(G)</b>, TEMPO <b>(H)</b>, or deferiprone <b>(I)</b> (n = 3). ^##<i>P</i> < 0.01 versus the control group; **<i>P</i> < 0.01 versus the Sph-treated group; ns, not significant. <b>(J)</b> Representative bright-field images of MAC-T cells following the indicated treatments. <b>(K-N)</b> Representative fluorescence images and (O-R) corresponding quantitative analyses of intracellular ROS (DCFH-DA; <b>K</b>, <b>O)</b>, Fe²⁺ accumulation <b>(L, P)</b>, lipid peroxidation (C11 BODIPY 581/591; M, <b>Q)</b>, and mitochondrial membrane potential (JC-10; N, R) in MAC-T cells treated with recombinant Sph (4 or 32 μg/mL) with or without deferiprone (30 μM) for 6 h (n = 3).