What is ferroptosis?
Ferroptosis is an iron-dependent form of programmed cell death with distinct morphological and biochemical characteristics that result in an increase in lipid peroxides. Morphologically, cells undergoing ferroptosis show reduced mitochondrial volume, increased bilayer membrane density, and reduction or disappearance of mitochondrial cristae. The cell membrane remains intact, the nucleus is normal in size, and there is no concentration of chromatin as seen in apoptosis. Similar to other regulated forms of necrosis, like necroptosis and pyroptosis, ferroptosis involves the loss of membrane integrity, but the mechanism of loss is different. The loss of membrane integrity is responsible for triggering an inflammatory response through the release of damage-associated molecular patterns (DAMPs).
This blog explores the key mechanisms, pathways, and proteins involved in ferroptosis, including the roles of iron homeostasis, lipid metabolism, and the regulation of oxidative stress.
Iron Homeostasis
Free ferrous iron (Fe2+) can lead to spontaneous lipid peroxidation through the Fenton reaction, which generates reactive oxygen species that damage cell membranes. Since ferroptosis is iron-dependent, it can be regulated by pathways that contribute to iron homeostasis and the availability of Fe2+. Notably, ferroptosis is inhibited by common iron chelators, such as deferoxamine.
Iron homeostasis involves multiple processes that control its absorption, transport, utilization, and storage. Ferric iron (Fe3+) is transported in the blood by the glycoprotein transferrin, which is produced in the liver. Iron is transported into cells upon binding of the transferrin-iron complex to the transferrin receptor (CD71, TFRC), a type II transmembrane receptor. Within endosomes in the cell, Fe3+ is reduced to ferrous iron by STEAP family members. Ferritin, which consists of heavy (FTH) and light (FTL) chains, is the major iron storage protein within cells, keeping iron in its non-toxic bioavailable form. Lysosomal-mediated degradation of ferritin, which can occur through a selective form of autophagy, termed ferritonphagy, can release iron and promote ferroptosis.
Immunohistochemical (IHC) analysis of paraffin-embedded human endometrioid adenocarcinoma using recombinant monoclonal antibody FTH1 (D1D4) Rabbit mAb #4393.
Ferritinphagy is mediated by a selective cargo receptor for ferritin, nuclear receptor coactivator 4 (NCOA4), that is targeted to the autophagosome. Alternatively, iron can be exported out of cells through ferroportin-1 (FPN1), a membrane protein that transports iron from inside the cell into the bloodstream.
Western blot (WB) analysis of extracts from various cell lines using recombinant monoclonal antibody NCOA4 (E8H8Z) Rabbit mAb #66849 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, HT-1080 and OVCAR-4 cells are low for NCOA4 expression.
Defense Against Oxidative Stress
Pathways regulating cellular defense against oxidative stress are critical to mitigate ferroptosis. Small-molecule lipophilic free radical scavengers, like ferrostatin-1 and liproxstatin-1, can inhibit ferroptosis. The glutathione pathway, in particular, has been identified as a key antioxidant defense pathway. A central player in this process is the metabolic protein glutathione peroxidase 4 (GPX4), which converts GSH into oxidized glutathione (GSSH), thus limiting cytotoxic lipid peroxidation and protecting cells against ferroptosis. The GPX4 inhibitor RSL3 is a potent inducer of ferroptosis.
IHC analysis of paraffin-embedded human papillary thyroid carcinoma using recombinant monoclonal antibody GPX4 (E5Y8K) Rabbit mAb #59735, a ferroptosis marker.
The glutathione peroxidase pathway is further regulated by System Xc-, an amino acid antiporter consisting of a disulfide-linked heterodimer of xCT/SLC7A11 and SLC3A2 (4F2hc/CD98). The System Xc- inhibitor erastin is a potent inducer of ferroptosis.
Immunofluorescent (IF) analysis of HeLa (left, high expressing) or SH-SY5Y (right, low expressing) cells using recombinant monoclonal antibody 4F2hc/SLC3A2 (D3F9D) XP® Rabbit mb #47213 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Another critical defense regulator is the transcription factor NRF2. NFR2 contributes to the regulation of genes involved in oxidative stress, including GPX4, and serves as a critical defense against ferroptosis.
Chromatin immunoprecipitations were performed with cross-linked chromatin from MEF cells treated with DEM (50 μM, 3 hr) and recombinant monoclonal antibody NRF2 (D1Z9C) XP® Rabbit mAb #12721 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 8 (upper), including NQO1 (lower), a known target gene of NRF2 (see additional figure containing ChIP-qPCR data).
Under normal conditions, the expression of NRF2 is inhibited through interaction with KEAP1, part of a ubiquitin E3 ligase complex that leads to NRF2 proteasomal degradation. During oxidative stress, KEAP1 undergoes conformational changes that disrupt this interaction, resulting in the stabilization of NRF2.
WB analysis of extracts from OVCAR8 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® KEAP1 siRNA I #5285 (+) or SignalSilence® KEAP1 siRNA II #5289 (+), using recombinant monoclonal antibody KEAP1 (D6B12) Rabbit mAb #8047 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The KEAP1 (D6B12) Rabbit mAb confirms silencing of KEAP1 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
This process is further regulated through the autophagy pathway, in which the autophagy cargo receptor SQSTM1/p62 can competitively inhibit the KEAP1-NRF2 complex, leading to the upregulation of NRF2.
IHC analysis of paraffin-embedded mouse forestomach using recombinant monoclonal antibody SQSTM1/p62 (D6M5X) Rabbit mAb #23214.
Lipid Metabolism
Lipid metabolism refers to the process of breaking down and synthesizing lipids. Lipids can contain either saturated fatty acids, which contain only single bonds between carbons, or unsaturated fatty acids, which include one or more double bonds between carbon atoms. Lipids containing polyunsaturated fatty acids (PUFAs) are vulnerable to peroxidation during ferroptosis.
ACSL4, an enzyme critical for lipid metabolism, is also a key regulator of ferroptosis. ACSL4 promotes the incorporation of PUFAs, such as arachidonic acid, into phospholipids, a process that enhances lipid peroxidation. High expression of ASCL4 leads to increased sensitivity to ferroptosis.
IF analysis of Caki-1 cells and MCF7 cells using ACSL4 (F6T3Z) Rabbit mAb #38493 (green), DyLight 554 Phalloidin #13054 (red), and DAPI #4083 (blue).
Antibody Sampler Kits & Tools for Studying Ferroptosis
Tools to monitor ferroptosis may involve multiple approaches, including pharmacological sensitivity (such as iron chelators and antioxidants), changes in expression of key targets (GPX4, SCL7A11, ferritin, NRF2, etc.), monitoring reactive oxygens and lipid peroxidation, and glutathione assays.
CST offers sampler kits to interrogate ferroptosis along with several other metabolic pathways to help you identify proteins relevant to your research and focus your initial efforts:
- Ferroptosis Antibody Sampler Kit #29650: This kit includes antibodies for key ferroptosis regulators such as GPX4, KEAP1, and NRF2. It's ideal for studying the molecular mechanisms of ferroptotic cell death and its regulation through pathways like the KEAP1-NRF2 axis.
- Redox/Ferroptosis Antibody Sampler Kit #70703: Intended for research into the relationship between redox regulation and ferroptotic processes, this kit includes antibodies for AIFM2/FSP1, GPX4, and thioredoxins.
- Iron Homeostasis/Ferroptosis Antibody Sampler Kit #36354: This kit includes antibodies against transferrin, transferrin receptor, and ferroportin-1, alongside ferroptosis-related markers like FTH1.
- Lipid Metabolism/Ferroptosis Antibody Sampler Kit #58434: For researchers investigating lipid remodeling and its impact on ferroptotic cell death, this kit includes antibodies for ACSL1, ACSL2, ACSL3, ACSL4, LPCATs, and SREBP-1.
- Glutathione Metabolism/Ferroptosis Antibody Sampler Kit #84600: This kit includes antibodies relevant for detecting proteins associated with glutathione metabolism and ferroptosis, including antibodies for GPX4, xCT/SLC7A11, GCLC, Glutaminase-1, Glutaminase-2, GOT1, and GOT2.
Additional Resources
To learn more about the mechanisms, morphology, and key proteins involved in many types of cell death, download the guide:
Read the additional blogs in the Mechanisms of Cell Death series:
- Mechanisms of Cell Death: Apoptosis
- Mechanisms of Cell Death: Necrosis & Necroptosis
- Mechanisms of Cell Death: Pyroptosis
Explore the CST® TUNEL kits, which robustly detect cells undergoing apoptosis and other forms of programmed cell death.
Select References
- Galluzzi L, Vitale I, Aaronson SA, et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 2018;25(3):486-541. doi:10.1038/s41418-017-0012-4
- Frank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019;26(1):99-114. doi:10.1038/s41418-018-0212-61.
- Escobar ML, Echeverría OM, Vázquez-Nin GH. Necrosis as Programmed Cell Death. In: Cell Death - Autophagy, Apoptosis and Necrosis. InTech.
- Yang WS, Stockwell BR. Ferroptosis: Death by Lipid Peroxidation. Trends Cell Biol. 2016;26(3):165-176. doi:10.1016/j.tcb.2015.10.014
Updated April 2025. Originally published June 2021. 20-CEP-94379 and 25-HMC-18550
