Fig 1: Downregulation of ALR increases Tfrc levels and cellular iron content through IRP1.Tfrc mRNA levels (A) and transferrin-dependent iron uptake (B) in wild type (WT) mouse embryonic fibroblasts (MEFs) with Alr downregulation (n = 5–6). (C) Steady-state 55Fe levels in cytosolic and mitochondrial fraction from cells treated with Alr siRNA (n = 6). (D) Schematic representation of Tfrc 3’UTR reporter constructs. (E) Full-length Tfrc 3’UTR reporter activities were measured in WT MEFs with ALR downregulation (n = 6). (F) IRE-deleted Tfrc 3’UTR reporter activities in WT MEFs with ALR downregulation (n = 14–18). (G) Alr and Tfrc mRNA levels in Aco1 knockdown (KD) MEFs treated with Alr siRNA (n = 4–6). (H) TFRC-mediated 55Fe uptake in Aco1 KD MEFs treated with Alr siRNA (n = 6). Experiments were run 48 hr after siRNA transfection for mRNA levels and 60 hr after transfection for steady state 55Fe levels and transferrin-mediated iron uptake. For luciferase assay, cells were sequentially transfected with siRNA and reporter construct (24 hr apart), and assayed 24 hr after transfection of reporter constructs. Data are presented as mean ± SEM. *p<0.05 by ANOVA. N.S. = not significant.
Fig 2: FPN1 protein level is decreased while its poly-ubiquitination is increased in the brain cortex of aged mice.(A) Immunoblots of iron transporting proteins TfR1 and FPN1 in the brain cortex of young and aged mice (n=3). (B) Summary of densitometric analysis of panel (A). (C) Poly-ubiquitination levels of FPN1, as assessed by immunoprecipitation, in the brain cortex of young and aged mice (n=3). (D) Summary of the densitometric analysis of panel (C). (E) Fe-S cluster containing aconitase enzyme activity, a marker of cellular oxidative stress, in the brain cortex of young and aged mice (n=4). c-aconitase = cytosolic aconitase (ACO1), m-aconitase = mitochondrial aconitase (ACO2). * p<0.05. Figure 3—source data 1.Full length images of immunoblots shown in Figure 3. Figure 3—source data 2.Densitometry – Original data of densitometry quantifications of immunoblots shown in Figure 3. Figure 3—source data 3.Aconitase assay – Original data of aconitase enzyme activities shown in Figure 3.
Fig 3: Elesclomol-copper eliminates pancreatic CSCs through induction of cuproptosis(A) Intracellular copper (Cu2+) content of human primary SIC002 PDAC cells after various treatments. Cells were cultured at 15,000 cells/well in 6-well ULA plates with 2 mL sphere culture medium, treated on day 3, and analyzed 4 days later using the Cell Copper (Cu) Colorimetric Assay Kit. (B) Western blot of Fe-S cluster proteins FDX1, LIAS, ACO-2, SDHB, and β-actin from extracts of SIC002 cells after various treatments. The western blots shown here were obtained from the same experiments as in Figure 2E. To ensure consistency, the membranes were stripped and reprobed as described in the materials and methods. Cells were cultured and treated as outlined in (A). (C) qPCR fold change of copper transporter-related genes ATP7A and ATP7B in SiC002 cells after various treatments. (D) Immunofluorescence imaging for DLAT (red) in SIC002 cells following allocated treatments. Mitochondria are stained with MitoTracker (green) and nuclei with DAPI (blue). (E) GSH level in SIC002 cells following allocated treatments. (F) Intracellular ROS levels in SIC002 cells following allocated treatments. (G and H) qPCR fold change of ROS-related genes NRF2 (G) and GCLC (H) in SIC002 cells following allocated treatments. (I and J) SOD1 activity (I) and aconitase activity (J) in SIC002 cells. For (A), (C), and (E)–(J), data are means ± SEM, with n = 3. ANOVA was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Please also see Figure S3.
Supplier Page from Abcam for Aconitase Assay Kit (Colorimetric)