Fig 2: IRP1/2 double-knockout cells expressing TFR1 can proliferate without iron supplementation and are sensitive to ferroptosis, but cannot sense ISC deficiency and enhance ferroptosis sensitivity.(A) Proliferation (relative cell number) of MDA-MB-231 cells expressing dox-repressible IRP2, TFR1 open reading frame (TFR1 without IRE) with 100 nM dox as indicated. (B) Immunoblots of IRP1/IRP2 double-knockout MDA-MB-231 cells expressing dox-repressible IRP2, TFR1 open reading frame, and nontargeting shRNA (shGFP, −) or shRNA targeting NFS1 (shNFS1, +), treated with 100 nM dox. (C) Relative viability of cells as in (B), treated with 100 nM dox, 1 μM ferrostatin, or erastin (0, 2.5, 5, and 10 μM) for 2 days as indicated. (D) Immunoblots of MDA-MB-231 cells expressing dox-repressible IRP2, TFR1 open reading frame, and shGFP or shRNAs targeting FXN (shFXN, 1 or 2), treated with 100 nM dox. (E) Relative viability of cells as in (D), treated with 100 nM dox, 50 μM Trolox, or erastin (0, 2.5, 5, and 10 μM) for 2 days as indicated. Asterisks indicate significance comparing shGFP to shNFS1 or shFXN for each group. P < 0.05, error bars are SEM.

Fig 3: IRP1 knockout cells activate the iron-starvation response upon inhibition of ISC synthesis and become sensitive to ferroptosis.(A) Relative viability of parental (WT) and IRP1 knockout MDA-MB-231 cell line expressing a nontargeting shRNA (shGFP, −) or NFS1 (shNFS1, +), treated with erastin (0, 2.5, 5, and 10 μM; 2 days). (B) Labile iron pool of parental and IRP1 knockout MDA-MB-231 cell line expressing shGFP or shNFS1. (C) Immunoblots for indicated proteins from lysates derived from parental (WT) and IRP1 knockout MDA-MB-231 cell line expressing shGFP or shNFS1, treated with 50 μM Deferoxamine (DFO) and 35 μM iron nitrate as indicated (1 day). (D) Relative abundance of TFR1 and FTH1 mRNA in parental and IRP1 knockout MDA-MB-231 cell line expressing shGFP or shNFS1. (E) Relative viability of WT and IRP1 knockout 293 human embryonic kidney (HEK) cell line expressing dox-inducible ISCU-DN treated with 100 nM dox (1 day) and then treated with 100 nM dox, 1 μM ferrostatin (Fer.), and erastin (Eras.; 0, 2.5, 5, and 10 μM; 2 days) as indicated. (F) Immunoblots for indicated proteins of lysates derived from parental and IRP1 knockout HEK-293 cell line expressing dox-inducible ISCU-DN treated with dox (0, 10, and 100 μM; 1 day) as indicated. (G) Immunoblots for indicated proteins of lysates derived from parental and IRP1 knockout MDA-MB-231 cells expressing GFP reporter with 5′UTR IRE sequence from FTH1 mRNA and shRFP or shNFS1. *P < 0.01; error bars are SEM.

Fig 4: Roles of T cell surface receptor molecules in Gal-3-triggered T cell apoptosis(A) Jurkat cells were transfected with siRNAs to CD45, CD29 or CD71 and compared with the negative control siRNA (siNC). The knockdown efficiency of CD45, CD29 and CD71 was determined by western blotting. (B) Jurkat cells from (A) were incubated with or without 2.5 μM Gal-3 (c refers to control). P-ERK1/2 and cleaved caspase-3 was determined by western blotting. (C-D) Control or CD45 knocked-down Jurkat cells were incubated with or without 2.5 μM Gal-3 and p-PKC was determined by western blotting (C) and ROS production was analyzed by flow cytometry (D). (E) Control or CD45 knocked-down Jurkat cells were incubated with or without 2.5 μM G69-250 variant and then analyzed for CD45, p-ERK, p-PKC and cleaved caspase-3. (F) Jurkat cell lysate was incubated with His-Gal-3- or His-G69-250-immobilized Ni-NTA beads. The upper panel shows CD45 determined by western blotting and the lower panel shows SDS-PAGE analysis of His-Gal-3 or His-G69-250 on the beads.

Fig 5: Iron-uptake and ferritinophagy-related gene expression are not affected by TP53 mutation status or erastin treatment. Relative mRNA expression of (A) transferrin receptor 1 (TFRC) (B) nuclear receptor coactivator 4 (NCOA4), (C) autophagy related 5 (ATG5), (D) CDGSG iron sulfur domain 1 (CISD1), and (E) solute carrier family 7 member 11 (SLC7A11) in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT) TP53, or the indicated TP53 mutant (n = 3 per cell type and treatment group). Relative mRNA expression of (F) TFRC, (G) NCOA4, (H) ATG5, (I) CISD1, and (J) SLC7A11 in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT) TP53, or the indicated mutant TP53 following treatment with DMSO (control) or 10 µM erastin for 24 h. a Denotes statistical difference from H1299. * Denotes statistical differences from respective control, p < 0.05. Error bars indicate SEM.