Fig 2: Activation and function of PTE in response to TNF. a APT1 and APT2 are part of the TNF-RSC: TNF-R1 was activated, lysed in the presence of detergents and precipitated using Fc60TNF, activating only TNF-R1. Panel one: TNF-R1 displaying the reported increase in K63-ubiquitination in response to TNF [5]. Panel two: Constitutive TNF-RSC:APT1 interaction, decreasing at 10 min. Panel three: Transient recruitment of APT2 to the TNF-RSC. Panels 4–6 show WB with 10 μg of input lysate. b Activation of endogenous S-depalmitoylases in response to TNF, analyzed by imaging flow cytometry using fluorescent probes: DPP-2 for global S-depalmitoylation and DPP-3 for APT1 activity. Left panel one: Representative images of inactive APT2. Left panel two: Shows cells with activated fluorescent probe (DPP-2; green) in response to TNF. PM is stained in red. Panel three: histograms representing enzyme activity. Dashed black: untreated cells. Green: addition of DPP-2 and TNF to cells kept on ice. Addition of DPP-2/TNF at 37 °C for 5 min (red), 10 min (orange), 15 min (blue). Black: DPP-2/Palmostatin B (pan PTE inhibitor). Dashed red: DPP-2/ML349 (APT2 selective inhibitor). Right panels: DPP-3 is not activated in response to TNF (same order and color code as A). c Left panel: representative WB from acylRAC probed for TNF-R1 at the conditions: no TNF (ctrl), 30 min TNF, ML349 with 30 min TNF, and ML349. PPT-1 serves as loading control. Right panel: WB quantification by densitometry. WB loading control, see Additional file 3: Figure S3. d His6rAPT2 was expressed in and affinity purified from E. coli. WB was probed using APT2 and His antibodies. rAPT2 activity after 20 min incubation at the conditions: DPP-2 (light grey), DPP-2 + APT2 (black), DPP-2 + APT2 + Palmostatin B (medium grey), DPP-2 + Palmostatin B (dark grey). e WB of input and acylRAC +/− rAPT2 (30 μM, 1 h 37 °C): Palmitoylation of TNF-R1 decreases while CD71, integrin α6, PPT-1 and Rab5 are not affected. The lower panel shows quantification of the WB for TNF-R1 normalized to PPT-1 levels

Fig 3: FTH-1 degradation is achieved by TetC-induced ferritophagy in BC cells. MCF7 and MDA-MB-231 cells were treated 20 and 10 μM TetC for 24 h in the presence or absence of 10 μM chloroquine (CQ). Western blot assay showed that TetC significantly elevated the expression of ATG5, ATG7, and NCOA4 but decreased FTH1 and TfR protein levels in MCF7 (A) and MDA-MB-231 cells (B). TetC-induced degradation of FTH-1 expression was obviously rescued by blocking the lysosome function with CQ in MCF7 (C) and MDA-MB-231 (D) cells. * p < 0.05; ** p < 0.01; *** p < 0.001 vs DMSO.

Fig 4: VPS34 inhibition depletes intracellular iron by blocking iron uptake through enhanced degradation of TFR and excess iron rescues the growth defect in RKO cells.(A) Aconitase activity in RKO cells upon VPS34 inhibition. RKO CTRL cells were treated with 1 μM PIK-III, 30 μM deferasirox (DFX) or vehicle control for 24 hours. The aconitase activity assay was used to quantify the isocitrate present in the cell extracts. All data was normalized to cells treated with vehicle, transformed to represent the percent isocitrate generated and averaged from three biological replicates ± SD; ****, p < 0.0001; NS, not significant (two-way ANOVA). (B) RKO CTRL cells were treated with vehicle or 0.5 μM PIK-III for 24 hours and lysates were immunoblotted with the indicated antibodies. (C) Quantitation of immunoblot in (B) as fold change versus vehicle treated cells. All data were normalized to the β-actin loading control. Data is averaged from three independent experiments ± SD, ****, p < 0.0001 (two-way ANOVA). (D) RKO.Cas9 cells were treated with vehicle or 1 μM PIK-III for 24 hours followed by incubation with Transferrin Alexa Fluor 488 probe for 30 min at 37°C. Nuclei were labeled with Hoechst and live cell imaging was performed. The white bar represents 10 μm in length. (E) Cellular uptake of Transferrin Alexa Fluor 488 probe from panel (D) was quantified as percent average intensity of total cells. Data were averaged and presented as the mean ± SD from 6 wells. (F) RKO CTRL cells were treated with the indicated concentrations of PIK-III for 1, 6, 12, or 24 hours and lysates were immunoblotted with the indicated antibodies. (G, H) RKO CTRL cells were treated with the indicated concentrations of PIK-III with or without 50 nM Bafilomycin A1 (G) or 30 μM chloroquine (H) for 12 and 24 hours and lysates were immunoblotted with the indicated antibodies. (I) RKO CTRL cells were treated with indicated concentrations of PIK-III along with vehicle, 1 μM or 50 μM Ferric Ammonium Citrate (FAC) for 5 days and cell viability was assessed using the CellTiter-Glo assay. Data is derived from three independent experiments and presented as mean ± SD, ***, p < 0.001; NS, not significant (two-way ANOVA). (J) RKO CTRL cells were treated with the indicated concentrations of PIK-III along with vehicle or 50 μM FAC for 24 hours. Lysates were immunoblotted with the specified antibodies. (K) Mitochondrial respiration defect due to PIK-III treatment. RKO CTRL cells were treated with the indicated concentrations of PIK-III along with vehicle for 24 hours with or without 50 μM FAC and mitochondrial respiration was assessed by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in response to the specified mitochondrial inhibitors using the Seahorse XFe96 analyzer. Oligomycin (Oligo) inhibits ATP synthase (Complex V) and decreases OCR; FCCP uncouples oxidative phosphorylation and increases OCR; and rotenone/antimycin A (ROT & AA) inhibits Complex I and III, respectively and decreases OCR. OCR and ECAR readouts from a single 96-well plate were normalized to the total DNA content measured in each well by DRAQ5 staining. Relative magnitude of mitochondrial oxidative phosphorylation and glycolysis is depicted as a ratio between the basal oxygen consumption rate and basal extracellular acidification rate (OCR/ECAR) and presented as the mean ± SD (n = 11 or 12 wells). Individual OCR and ECAR graphs are shown in S5 Fig.

Fig 5: Expression of salusin-β during the process of ferroptosis. (A) HK-2 cells were treated with ferroptosis activators for 24 h, including erastin (5 µM), GPX4 inhibitors (1 µM for RSL3 and 5 µM for FIN56) and GSH synthase inhibitor (100 µM for buthionine sulfoximine), the protein expression of salusin-β was analyzed by western blotting. (B) HK-2 cells were pretreated with ferrostatin-1 (1 µM) for 30 min and incubated with HG for 48 h, the protein expression of salusin-β was then detected by western blotting. (C) The action mechanism of salusin-β involved in ferroptosis in diabetic kidneys via inactivation of Nrf-2 signaling. Data are presented as the mean ± SD (n=5 per group). *P<0.05 vs. Veh or NG. †P<0.05 vs. HG. GPX4, glutathione peroxidase 4; GSH, glutathione; Nrf-2, nuclear factor erythroid-derived 2-like 2; NG, normal glucose; HG, high glucose; Veh, Vehicle SLC7A11, solute carrier family 7 (cationic amino acid transporter, y+ system) member 11; FTH-1, ferritin heavy polypeptide 1; TFR-1, transferrin receptor 1; ROS, reactive oxygen species.