Fig 2: Ferritinophagy mediates ferroptotic tumor suppression.A Western blot analysis of protein expression in control and ATG5-knockdown Y79-CR cells following treatment with erastin (10 µM) for 24 h. B Analysis of intracellular free iron in control and ATG5-knockdown Y79-CR cells following treatment with erastin (10 µM) for 8–24 h (two-way ANOVA with Tukey’s multiple comparisons test; data are presented as mean ± SD; n = 3 biologically independent samples). C Western blot analysis of protein expression in control and NCOA4-knockdown Y79-CR cells following treatment with erastin (10 µM) for 24 h. D Analysis of intracellular free iron in control and NCOA4-knockdown Y79-CR cells following treatment with erastin (10 µM) for 8–24 h (two-way ANOVA with Tukey’s multiple comparisons test; data are presented as mean ± SD; n = 3 biologically independent samples). E–H Indicated Y79-CR cells were treated with erastin (5–10 µM) for 24 h, and then cell viability (E), cell death (F), HMGB1 release (G), and lipid ROS (H) were assayed (two-way ANOVA with Tukey’s multiple comparisons test; data are presented as mean ± SD; n = 3 biologically independent samples).

Fig 3: Schematic description of GCH1/BH4 metabolism suppression of erastin-induced ferroptosis by reducing lipid peroxidation and silencing NCOA4-mediated ferritinophagy in CRC. DAHP, an inhibitor of GCH1; TFR1, transferrin receptor 1; TF, transferrin; DMT1, divalent metal transporter 1; GSH, Glutathione; GPX4, Glutathione peroxidase 4; GCH1, GTP cyclohydrolase-1; BH4, tetrahydrobiopterin; PTPS, 6-pyruvoyl tetrahydropterin synthase; SR, sepiapterin reductase; Mito, mitochondria.

Fig 4: Effects of the mycalol mixture on ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and nuclear receptor coactivator 4 (NCOA4). Western blotting analyses with anti-GPX4 (a) or anti-NCOA4 (b) antibody of the extracts from HepG2 treated with the DMSO alone (control) or in the presence of mycalols. The intensity of the bands of GPX4 (c) and NCOA4 (d) were normalized, respectively, with β-tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as standard proteins, and are reported as relative to GPX4 and NCOA4 levels (* p < 0.05; ** p < 0.01).

Fig 5: Schematic summary of targeting autophagy for RB therapy.Persistent cells are a small subgroup of cancer cells that remain viable under carboplatin treatment, leading to multidrug resistance by increasing autophagy activity. Up-regulated autophagy inhibits caspase-mediated apoptosis, but increases the sensitivity of drug-resistant RB cells to ferroptosis. In particular, 4-octyl itaconate activates NCOA4-mediated ferritinophagy, leading to ferritin degradation and subsequent free iron accumulation, and finally oxidative damage through the iron-mediated ROS production via Fenton reaction.