Fig 2: Suppression of ISC synthesis increases IRP2-mRNA binding independent of altered IRP2 stability at tissue O2 levels.(A and B) EMSA using 32P-labeled IRE probe (A), or immunoblot for indicated proteins (B), of lysates derived from parental and IRP1 knockout HEK-293 cell line expressing dox-inducible ISCU-DN, treated with 100 nM dox (1 day), and incubated in 21% O2, lysis in 1% O2. (C and D) EMSA using 32P-labeled IRE probe (C) and immunoblot for indicated proteins (D) of lysates derived from parental and IRP1 knockout MDA-MB-231 cell line expressing shGFP or shNFS1, incubated in 3% O2, lysis in 1% O2.

Fig 3: PEITC altered iron metabolism in human OS cells. (a) Total iron level in MNNG/HOS, U-2 OS, MG-63, and 143B cells after the indicated concentrations of PEITC treatment for 24 h by AAS. (b) The level of labile iron in MNNG/HOS, U-2 OS, MG-63, and 143B cells after the indicated concentrations of PEITC treatment for 24 h with Calcein-AM staining by flow cytometry analysis. (c) Protein expression levels of TfR1, DMT1, FTH1, FPN, and IRP2 in MNNG/HOS, U-2 OS, MG-63, and 143B cells treated with the indicated concentrations of PEITC for 20 h or 30 μM PEITC for 4 h, 12 h, 24 h, and 48 h. (d) mRNA levels of TFRC, SLC11A2, FTH1, SLC40A1, and IRP2 in MNNG/HOS, U-2 OS, MG-63, and 143B cells after PEITC treatment for 24 h. (e) Protein expression levels of NCOA4 in MNNG/HOS, U-2 OS, MG-63, and 143B cells treated with the indicated concentrations of PEITC for 20 h or 30 μM PEITC for 4 h, 12 h, 24 h, and 48 h. All data were presented as the means ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus control group.

Fig 4: NCOA4-mediated ferritinophagy supports ISC protein levels and activity in PDAC cells. A, Network map showing gene dependencies most highly correlated with NCOA4 dependency in the DepMap (21Q1). Gray connections indicate the interaction between genes as predicted by STRING. Functional clusters manually annotated based on Gene Ontology (GO) biological process analysis as in B. FBXL5 is anticorrelated to NCOA4 dependency. B, GO biological process enrichment analysis of gene dependencies most highly correlated with NCOA4 dependency in the DepMap (21Q1). LDL, low-density lipoprotein. C, Volcano plot illustrates statistically significant protein abundance differences in TCC-Pan2 cells lentivirally transduced with sgNCOA4-1–Cas9 versus sgControl–Cas9. Volcano plots display the −log10 (P) versus the log2 of the relative protein abundance of mean sgNCOA4-1 to mean sgControl samples. Orange circles represent ISC proteins identified in the data set (n = 53). Red circles represent significantly upregulated proteins (log2 fold change >0.5), whereas blue circles represent significantly downregulated proteins (log2 fold change <−0.5; data from 4 sgControl or 3 sgNCOA4-1 independent plates). D, Enrichment map of GSEA-Reactome of sgNCOA4-1 compared with sgControl TCC-Pan2 proteome (P < 0.01; FDR q value <0.1; Jaccard coefficient >0.5). Node size is related to the number of components identified within a gene set, and the width of the line is proportional to the overlap between related gene sets. GSEA terms associated with upregulated (red) and downregulated (blue) proteins are colored accordingly and grouped into nodes with associated terms. ECM, extracellular matrix; ER, endoplasmic reticulum. E, Enrichment plot for GO (molecular function) Metal Cluster Binding gene set of sgNCOA4-1 compared with the sgControl TCC-Pan2 proteome. F, Log2 (fold change) heat map for ISC-containing proteins and ISC synthesis proteins as measured from TCC-Pan2 cells [comparisons are sgNCOA4-1/sgControl, sgNCOA4–2/sgControl, sgControl + deferasirox (DFX)/sgControl]. Values presented are the mean of 3 independent plates. Color legends: color scheme for functional and localization categories of ISCs. TCA, tricarboxylic acid. G, Immunoblot showing ISC protein levels in lysates from TCC-Pan2 and PANC-1 cell lines lentivirally transduced with sgControl, sgNCOA4-1, or sgNCOA4-2 (NCOA4 KD). Relative fold-change quantification is of 2 to 3 replicates. Increased IREB2 levels serve as a positive control indicative of decreased free iron. H, Immunoblot showing ISC protein levels in lysates from TCC-Pan2 cell lines lentivirally transduced with sgControl or sgNCOA4-1 with or without FAC (100 μmol/L). Relative fold-change quantification is of 4 replicates. I, FECH activity from TCC-Pan2 cells lentivirally transduced with sgControl, sgNCOA4-1, or sgControl cells treated with DFO (12 hours, 1 mmol/L) as measured by the formation of zinc–mesoporphyrin (monitored at 400 nm). J, Cytosolic aconitase activity from TCC-Pan2 cells lentivirally transduced with sgControl, sgNCOA4-1, or sgControl cells treated with DFO (12 hours, 1 mmol/L). K, OCR of mitochondrial respiratory complex using Seahorse in TCC-Pan2 cells transduced with siControl or siNCOA4. L, Maximal OCR in TCC-Pan2 cells transduced with siControl or siNCOA4. For G–J and L, significance was determined with the t test. ns = not significant: P > 0.1; ^, P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars, ± SD.

Fig 5: CISD2 silencing accelerates iron accumulation in A549 cells. (A) The activity of cytoplasmic aconitase in CISD2 knockdown cells, **P < 0.01 vs. the control group. (B) The activity of mitochondrial aconitase in CISD2 knockdown cells, *P < 0.05 vs. the control group. (C) Protein levels of iron regulatory protein 2 (IRP2), transferrin receptor protein (TFR), and hypoxia-inducible factor 1-alpha (HIF1-α) in indicated A549 cells. β-Actin was used as the loading control. (D) Intracellular Fe2+ was measured by RPA staining and flow cytometry analysis. Representative images (left) and statistical graphs (right) are shown; *P < 0.05, compared to the control group. (E) The representative images of mitochondrial morphology. Control and CISD2 silenced A549 cells were treated with or without DFO (100 μM) for 12 h, then labeled with MitoTracker Red and subjected to a confocal microscope for the observation of changes in mitochondrial morphology. Scale bars: 5 µm. (F) Quantification of mitochondrial morphology alteration according to the classification of mitochondrial category I–III. (G) The morphological changes of mitochondria in control and CISD2 silenced cells were detected by transmission electron microscopy (TEM) in the presence or absence of DFO (100 μM) treatment. Comparable results were obtained in three independent experiments.