Ferroptosis: Mechanisms in Disease and Kit Selection DOJINDO LABORATORIES

Science Note

New Insights into GPX4 Control of Ferroptosis [Jan. 6, 2026]

Previous Science Note

GPX4 is a central suppressor of ferroptosis that reduces peroxidized phospholipids within cellular membranes, and its regulation is closely linked to the prevention of neurodegenerative diseases and the development of cancer therapies. Accordingly, new insights into GPX4 biology are important for advancing our understanding of ferroptosis control. Recent studies have shown that mutations disrupting the membrane-anchoring mechanism of GPX4 cause its mislocalization, which is sufficient to induce ferroptosis and neurodegeneration independently of enzymatic activity. In addition, PRDX6 has been identified as a selenium-acceptor protein required for GPX4 synthesis, and loss of PRDX6 leads to reduced GPX4 abundance and increased sensitivity to ferroptosis. Together, these findings provide an important foundation for modulating ferroptosis through GPX4 regulation and translating this knowledge into therapeutic applications.

1. A fin-loop-like structure in GPX4 underlies neuroprotection from ferroptosis (Cell, 2025)
Summary: GPX4 is a key enzyme in suppressing ferroptosis, but the importance of its subcellular localization has not been fully elucidated. Using GPX4 variants carrying mutations in the membrane-anchoring fin-loop, this study demonstrates across human iPSC-derived models, organoids, and mice that disruption of this anchoring mechanism is sufficient to trigger ferroptosis, and that GPX4 mislocalization rather than loss of enzymatic activity alone directly underlies ferroptosis-driven neurodegeneration.

Highlighted technique: To evaluate the importance of GPX4 localization, patient-derived primary fibroblasts carrying the R152H mutation in the membrane-anchoring fin-loop of GPX4 were used to analyze the impact of anchor disruption. Lipid peroxidation was assessed using BODIPY 581/591 C11, demonstrating that the R152H mutant exhibits increased accumulation of peroxidized lipids.

2. PRDX6 dictates ferroptosis sensitivity by directing cellular selenium utilization (Molecular Cell, 2024)
Summary: GPX4 is a selenium-dependent phospholipid hydroperoxide reductase and the central suppressor of ferroptosis, whose proper protein expression requires efficient selenium incorporation. Although PRDX6 itself does not directly suppress ferroptosis, this study demonstrates that PRDX6 acts as a selenium-acceptor protein essential for GPX4 synthesis, such that loss of PRDX6 reduces GPX4 abundance and increases ferroptosis sensitivity.

Highlighted technique: Using PRDX6 KO cells, this study shows that loss of PRDX6 markedly reduces GPX4 protein levels without altering GPX4 mRNA expression, demonstrating that PRDX6 is required for GPX4 synthesis rather than transcription. Consistently,PRDX6-deficient mice exhibit a selective reduction of GPX4 expression in the brain, where selenium supply and utilization are physiologically constrained, highlighting the essential role of PRDX6 in maintaining GPX4 abundance under selenium-limited conditions.

Ferroptosis Indicators (click to open/close)

Target	Kit & Probes
Lysosomal ferrous ion (Fe²⁺) detection	Lyso-FerroRed ^NEW
Ferroptosis Indicator: ferrous ion (Fe²⁺)	FerroOrange(intracellular), Mito-FerroGreen(mitochondria)
Ferroptosis Indicator: lipid peroxidation	Liperfluo(intracellular), MitoPeDPP(mitochondria)
Lipid Peroxidation Assay	Lipid Peroxidation Probe -BDP 581/591 C11-
Malondialdehyde Detection	MDA Assay Kit
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Total ROS detection	Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Glutathione Quantification	GSSG/GSH Quantification Kit
Cystine Uptake detection	Cystine Uptake Assay Kit

Application Note (click to open/close)
> When Lysosomes Go Neutral: Iron Loss Unveiled

We investigated the transition of cellular metabolisms in A549 cells treated with erastin, a known ferroptosis inducer. Our results revealed the following.

Results
- The inhibition of cystine uptake by erastin led to a depletion of cysteine, which in turn increased the compensatory uptake of other amino acids.
- Glucose uptake, which typically promotes ferroptosis*, was found to decrease upon erastin treatment, suggesting a potential cellular self-defense mechanism.
- The depletion of cysteine resulted in a decrease in glutathione levels and an increase in Fe²⁺, ROS, and lipid peroxides, all of which are recognized markers of ferroptosis.

Cell Line: A549
Incubation Conditions: 100 μmol/l Erastin/MEM, 37℃, 3h
*Reference: Xinxin Song, et al., Cell Reports, (2021)

Products in Use
① Amino Acid Uptake: Amino Acid Uptake Assay Kit
② Glucose Uptake: Glucose Uptake Assay Kit-Green
③ Cystine Uptake: Cystine Uptake Assay Kit
④ Intracellular glutathione: GSSG/GSH Quantification Kit
⑤ Intracellular labile Fe: FerroOrange
⑥ Intracellular total ROS: ROS Assay Kit -Highly Sensitive DCFH-DA-
⑦ Lipid Peroxides: Liperfluo

Why is ferroptosis research important?

Detection of ferroptosis is essential to elucidate its impact on neurodegenerative diseases and cancer, as it plays a role in neuronal loss in diseases such as Alzheimer's and Parkinson's, while also representing a potential therapeutic target in malignancies. Reliable detection of ferroptosis supports the development of neuroprotective strategies to slow disease progression and improves cancer treatment approaches by promoting ferroptotic cell death in therapy-resistant tumors.

Master the Basics with an Overview Map!

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Topics

What is Ferroptosis?
How Does Ferroptosis Cause Cell Death?
Research on Related Diseases
Ferroptosis-Related Reagent Selection Guide
Experimental Example: Evaluating intracellular uptake and redox balance in erastin-induced ferroptosis
Experimental Example: Changes in various indicators of cell death induced by drugs

What is Ferroptosis?

“Ferroptosis” was coined by Stockwell et al. at Columbia University in 2012 and described as a form of iron-dependent cell death. * It was reported to be a form of programmed cell death by the Nomenclature Committee on Cell Death (NCCD) in 2018.
Ferroptosis is a form of programmed cell death caused by iron ion-dependent accumulation of lipid peroxides. Ferroptosis has been shown to follow a different cell death pathway from apoptosis and thus is attracting attention as a new target for cancer therapy. It has also been found to be associated with various diseases, such as neurodegenerative diseases, cerebral apoplexy, and hepatitis (MASH).

*S. J. Dixon, B. R. Stockwell, et al., Ferroptosis: an iron-dependent form of nonapoptotic cell death., Cell, 2012, 149(5), 1060.

How Does Ferroptosis Cause Cell Death?

Ferroptosis is characterized by the accumulation of lipid peroxides. Lipid peroxides are formed from oxidation of polyunsaturated fatty acids (PUFA) in membrane phospholipids, with iron suggested to be involved. Intracellular glutathione peroxidase 4 (GPX4) uses reduced glutathione (GSH), an antioxidant, to reduce lipid peroxides generated by reactive oxygen species (ROS).*
However, when lipid peroxides accumulate due to GPX4 disruption or GSH depletion, ferroptosis is triggered.

*Stockwell et al, a leading researcher in the field of ferroptosis, summarized inhibitors, inducers, and detection indicators of ferroptosis in the following review, in which Dojindo’s Liperfluo is introduced for detection of lipid peroxides.

B. R. Stockwell, et al., "Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease.", Cell, 2017, 171, 273.

Research on Related Diseases

Metabolic dysfunction-associated steatohepatitis (MASH)
Suppression of hepatitis via ferroptosis In a study involving the livers of MASH model mice, it was confirmed that necrosis precedes apoptosis in the development of fatty liver. Further experiments showed that ferroptosis is involved within necrosis as a trigger for steatohepatitis and that inhibition of ferroptosis almost completely suppressed the onset of hepatitis. Minoru Tanaka, et al., "Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis", Cell Death & Disease, 2019, 10, 449.
Related article: changes in intracellular markers associated with MASH The article summarizes reports on changes in each indicator of metabolic states and cellular senescence using the NASH model. (Click on the “MASH” tab in the link) Experimental example: measurement of intracellular metabolism in MASH model tissue Measurement of ATP, a-KG, and NAD levels in liver tissue of high-fat diet-treated type 1 diabetic model mice. (Please refer to each product’s website for more information, “Experimental Example: Change in Metabolism in Liver Tissue of MASH-Induced Mouse”)

Metabolic dysfunction-associated steatohepatitis (MASH)

Suppression of hepatitis via ferroptosis

In a study involving the livers of MASH model mice, it was confirmed that necrosis precedes apoptosis in the development of fatty liver. Further experiments showed that ferroptosis is involved within necrosis as a trigger for steatohepatitis and that inhibition of ferroptosis almost completely suppressed the onset of hepatitis.

Minoru Tanaka, et al., "Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis", Cell Death & Disease, 2019, 10, 449.

Related article: changes in intracellular markers associated with MASH

The article summarizes reports on changes in each indicator of metabolic states and cellular senescence using the NASH model.

(Click on the “MASH” tab in the link)

Experimental example: measurement of intracellular metabolism in MASH model tissue

Measurement of ATP, a-KG, and NAD levels in liver tissue of high-fat diet-treated type 1 diabetic model mice. (Please refer to each product’s website for more information, “Experimental Example: Change in Metabolism in Liver Tissue of MASH-Induced Mouse”)

Neurodegenerative disease
Confirmation of the link between lysosomal disorders and ferroptosis In experiments using human neurons, it is reported that knockdown of the lysosomal protein prosaposin induces formation of lipofuscin, a hallmark of aging. This process involves the iron-catalyzed generation of reactive oxygen species, leading to induction of ferroptosis. Martin Kampmann, et al., "Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis", Nature Neuroscience, 2021, 24, 1020

Neurodegenerative disease

Confirmation of the link between lysosomal disorders and ferroptosis

In experiments using human neurons, it is reported that knockdown of the lysosomal protein prosaposin induces formation of lipofuscin, a hallmark of aging. This process involves the iron-catalyzed generation of reactive oxygen species, leading to induction of ferroptosis.

Martin Kampmann, et al., "Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis", Nature Neuroscience, 2021, 24, 1020

Cancer
Regulation of cancer immunity via ferroptosis CD8+ T cells activated by immunotherapy were found to confer an anti-tumor effect by promoting lipid peroxidation and inducing ferroptosis. The mechanism of immunotherapy-induced inhibition of cystine uptake and promotion of lipid peroxidation in tumor cells is discussed. Weiping Zou, et al, "CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy", Nature, 2019, 569, 270

Cancer

Regulation of cancer immunity via ferroptosis

CD8+ T cells activated by immunotherapy were found to confer an anti-tumor effect by promoting lipid peroxidation and inducing ferroptosis. The mechanism of immunotherapy-induced inhibition of cystine uptake and promotion of lipid peroxidation in tumor cells is discussed.

Weiping Zou, et al, "CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy", Nature, 2019, 569, 270

Ferroptosis-Related Reagent Selection Guide

Lipid Peroxide and Iron (Fe²⁺) Detection Reagents

Name	Liperfluo	MitoPeDPP	Lipid Peroxidation Probe -BDP 581/591 C11-	MDA Assay Kit	Mito-FerroGreen	FerroOrange
Target	Lipid Peroxide	Lipid Peroxidation	Lipid Peroxidation	Malondialdehyde	Ferrous Ion(Fe²⁺)	Ferrous Ion(Fe²⁺)
Localization	Intracellular	Mitochondria	Intracellular	Intracellular	Mitochondria	Intracellular
Detection (Fluorescence:Ex/Em)	Fluorescence (524 nm/535 nm)	Fluorescence (452 nm/470 nm)	Fluorescence 1. 488 nm/510-550nm 2. 561 nm/600-630nm	Fluorescence (540 nm/590 nm) Colorimetric: 532 nm	Fluorescence (505 nm/580 nm)	Fluorescence (543 nm/580 nm)
Instrument	Fluorescence Microscope, FCM	Fluorescence Microscope, FCM	Fluorescence Microscope, FCM, Microplate Reader	Microplate Reader	Fluorescence Microscope, Microplate Reader	Fluorescence Microscope
Sample	Live Cell	Live Cell	Live Cell	Cell, Tissue	Live Cell	Live Cell

Oxidative Stress- and Metabolism-Related Reagents and Kits

Name	ROS Assay Kit -Highly Sensitive DCFH-DA-	GSSG/GSH Quantification Kit	Glutamine Assay Kit-WST	Glutamate Assay Kit-WST
Target	ROS (Reactive oxygen species)	Glutathione (oxidized/reduced)	Glutamine	Glutamine
Localization	Intracellular	Intracellular	Intracellular/Extracellular	Intracellular/Extracellular
Detection (Fluorescence:Ex/Em)	Fluorescence (505 nm/525 nm)	Colorimetric:412 nm	Colorimetric:450 nm	Colorimetric:450 nm
Instrument	Fluorescence Microscope, FCM, Microplate Reader	Microplate Reader	Microplate Reader	Microplate Reader
Sample	Live Cell	Cell, Tissue, Blood Plasma, Red Blood Cell	Cell, Culture Medium	Cell, Culture Medium

Experimental Example: Evaluating intracellular uptake and redox balance in erastin-induced ferroptosis

We investigated the transition of cellular metabolisms in A549 cells treated with erastin, a known ferroptosis inducer. Our results revealed the following.

Results
- The inhibition of cystine uptake by erastin led to a depletion of cysteine, which in turn increased the compensatory uptake of other amino acids.
- Glucose uptake, which typically promotes ferroptosis*, was found to decrease upon erastin treatment, suggesting a potential cellular self-defense mechanism.
- The depletion of cysteine resulted in a decrease in glutathione levels and an increase in Fe²⁺, ROS, and lipid peroxides, all of which are recognized markers of ferroptosis.

Cell Line: A549
Incubation Conditions: 100 μmol/l Erastin/MEM, 37℃, 3h
*Reference: Xinxin Song, et al., Cell Reports, (2021)

Products in Use
① Amino Acid Uptake: Amino Acid Uptake Assay Kit
② Glucose Uptake: Glucose Uptake Assay Kit-Green
③ Cystine Uptake: Cystine Uptake Assay Kit
④ Intracellular glutathione: GSSG/GSH Quantification Kit
⑤ Intracellular labile Fe: FerroOrange
⑥ Intracellular total ROS: ROS Assay Kit -Highly Sensitive DCFH-DA-
⑦ Lipid Peroxides: Liperfluo

Experimental example: Changes in various indicators of cell death induced by drugs

HepG2 cells treated with the apoptosis-inducing agent staurosporine or the ferroptosis-inducing agents Erastin and RSL3. After treatment, extracellular LDH, phosphatidylserine, cell viability, intracellular Fe²⁺ and lipid peroxidation were determined.
The results showed that apoptosis-induced cells treated with staurosporine showed an increase in phosphatidylserine, a decrease in cell viability and an increase in extracellular LDH, indicating that cell death had occurred. On the other hand, intracellular Fe²⁺, an indicator of ferroptosis, remained unchanged. In cells treated with Erastin, a ferroptosis inducer, intracellular Fe²⁺ increased and cell viability decreased, but extracellular LDH and lipid peroxidation (lipid peroxidation: decrease in red fluorescence and increase in green fluorescence) did not increase. In cells in which ferroptosis was more strongly induced by co-treatment with RSL3 in addition to Erastin, increased intracellular Fe²⁺ and lipid peroxidation were observed. Moreover, decreased cell viability and increased dead cells were detected. Meanwhile, phosphatidylserine showed a lower rate of increase during ferroptosis induction compared to apoptosis-induced cells. These results suggest that cell death can be distinguished by evaluating a combination of cell death indicators.

[Products in use]
Extracellular LDH : Cytotoxicity LDH Assay Kit-WST (Product code: CK12)
Phosphatidylserine: Annexin V Apoptosis Plate Assay Kit（Product code: AD12）
Cell viability : Cell Counting Kit-8 (Product code: CK04)
Intracellular Fe²⁺ : FerroOrange (Product cose: F374) *Normalized with Hoechst 33342 fluorescence intensity
Lipid peroxidation : Lipid Peroxidation Probe -BDP 581/591 C11- (Product code: L267)

[Experimental conditions]
Cell type: HepG2 cell(2×10⁴ cells/well)
Drugs: Staurosporin(5 μmol/l), Erastin(25 µmol/l), Erastin+RSL3(both 25 µmol/l) *Diluted in serum-free medium