The A431 cell line is a human epidermoid (squamous cell) carcinoma line prized for one property above all others — an unusually high surface density of epidermal growth factor receptor (EGFR) — which makes it the default signal ceiling for calibrating EGFR-directed binders and probes. But of the 37 publications associated with this catalog item, only five are primarily EGFR studies; the rest use A431 as a malignant keratinocyte counterpart, a tumor-forming xenograft substrate, a cytotoxicity screen, or a human epithelial background. This application note takes one representative study from each of the five best-attested application areas and describes what researchers actually did with the cells: the comparator, the readout, and the result.
What the A431 cell line is, and why it is cited
A431 was established in 1973 by Giard and colleagues from an epidermoid carcinoma in an 85-year-old female patient. It grows as an adherent monolayer of flat, polygonal, epithelial-like cells. The receptor is why it is stocked; the squamous phenotype is why it is cited. One mechanistic detail underpins several of the studies below: EGF stimulation of A431 produces rapid tyrosine phosphorylation of membrane proteins and engages MAPK/ERK and PI3K/AKT signaling, but the response is biphasic — low ligand concentrations drive proliferation, while high concentrations inhibit growth and push cells toward terminal differentiation. A single model therefore yields two opposing readouts depending on dose. A431 is available in the BioHippo cell lines catalog.
Five representative studies: what researchers did with A431 cells
Each card covers one study in depth, read against the primary article. The cluster label gives the application area and how many studies in the reference list belong to it.
Resveratrol reduced EGFR phosphorylation at specific tyrosine residues; its methylthio analogues did not improve on it
Cichocki M, Szaefer H, Krajka-Kuźniak V, Baer-Dubowska W. Mol Cell Biochem 2014;396(1–2):221–8. DOI · PMID 25063218
A431's roleThe malignant arm of a two-line design. A431 (epidermoid carcinoma) was run in parallel with HaCaT immortalized keratinocytes so the same compounds could be assessed in a high-EGFR carcinoma context and a non-malignant one.
The team tested resveratrol against two 4′-methylthio-trans-stilbene derivatives, asking whether methylthio substitution produces a better modulator of the EGFR/Stat3 axis. Stat3 activation was measured as the amount of Stat3 recovered in DNA-binding complexes from nuclear extracts.
- In A431, resveratrol at the higher dose significantly reduced phosphorylation at both Y1173 and Y1068 of EGFR (p < 0.05); S2 affected only Y1068.
- In A431, resveratrol and S2 significantly diminished Stat3 DNA-binding capacity; the same compounds barely moved Stat3 activation in HaCaT.
- In HaCaT, resveratrol and both derivatives lowered total EGFR protein — a different mode of action from the residue-specific dephosphorylation seen in A431.
- The authors concluded the methylthiostilbenes are not more potent than resveratrol.
Why this is a useful templateThe readout is residue-resolved rather than a bulk viability number — which is what makes A431's receptor abundance worth paying for. The HaCaT pairing is what allows the (negative) conclusion to mean anything.
A431 was used as a TP53R273H-bearing line to show that mutant p53 binds BCAR1 and drives invasion
Guo AK, Itahana Y, Seshachalam VP, Chow HY, Ghosh S, Itahana K. Br J Cancer 2021;124(1):299–312. DOI · PMID 33144694
A431's roleAn endogenous TP53R273H background. A431 carries this hotspot mutant natively, so it served as a physiologically relevant setting for siRNA knockdown (40 nM against TP53 or BCAR1) alongside MDA-MB-468, and against the p53-null H1299 line used for reconstitution.
Using immunoprecipitation and proximity ligation assays, the group identified BCAR1 (p130Cas) as a novel TP53R273H binding protein, then tested the functional consequence by transwell invasion assay.
- BCAR1 translocates from cytoplasm to nucleus and binds TP53R273H in a manner dependent on SRC family kinases.
- Full-length TP53R273H promoted invasion; the binding-deficient deletion mutant did not — tying the phenotype to the interaction, not to mutant p53 generally.
- In patients with mutant TP53, high BCAR1 expression was associated with poorer prognosis (a correlative association, not a validated stratifier).
Correction worth carrying forwardSupplier records for A431 commonly state only “p53 positive.” The protein present is the R273H hotspot mutant, a DNA-contact mutation with documented gain-of-function behaviour. If your work touches p53, treat A431 as a mutant-p53 model — and confirm the allele in your own lot.
Unstimulated A431 already carried higher antioxidant levels than HaCaT — which is why plasma affected the two lines differently
Manzhula K, Rebl A, Budde-Sagert K, Rebl H. Int J Mol Sci 2024;25(20):10967. DOI · PMID 39456749
A431's roleThe squamous cell carcinoma arm, cultured under conditions identical to HaCaT so baseline redox differences could be attributed to the cells rather than the medium.
The group quantified NF-κB, IκBα, Nrf2, Keap1, IKK and phospho-IKK, and measured nuclear translocation after cold atmospheric pressure plasma (CAP) exposure of up to 180 s.
- At baseline the two lines had similar ROS levels but significantly higher antioxidant levels in A431.
- CAP raised antioxidant capacity in HaCaT and drove strong Nrf2 nuclear translocation there, upregulating HMOX1 and GPX1.
- A431 showed only a modest Nrf2 increase (~+48%) — limited headroom for further redox adaptation.
- CAP selectively induced NF-κB activation in A431 while total NF-κB fell, and downregulated IL1B, IL6 and CXCL8.
Design implicationThe selectivity is not that A431 is intrinsically more fragile — it has less remaining capacity to adapt. Measure baseline antioxidant status before treatment, or an apparent selectivity effect may simply be a difference in starting reserve.
A431 xenografts supplied the highly angiogenic reference phenotype for a super-resolution ultrasound method
Opacic T, Dencks S, Theek B, et al. Nat Commun 2018;9:1527. DOI · PMID 29670096
A431's roleOne of three xenograft models chosen for contrasting vascular phenotypes — A431 (epidermoid), MLS (ovarian) and A549 (lung) — with 4 × 106 cells injected subcutaneously into female CD1-nude mice, n = 4 per model. A431 provided the highly angiogenic end of the spectrum.
The paper introduces motion model ultrasound localization microscopy (mULM), reaching ~10 µm resolution in 40 s on a conventional 50 fps preclinical scanner, cross-validated against µCT of Microfil-perfused tumors and CD31 immunohistochemistry.
- A431 tumors showed a fine, homogeneously distributed microvessel network and the highest relative blood volume of the three models.
- A431 had the smallest distance to the nearest vessel, by both mULM and IHC.
- Two parameters classified all three models correctly (100%); A431 vs. MLS and A431 vs. A549 were both significant at p < 0.01.
Note on provenance and mediumThis study sourced A431 as catalog no. 300112 from Cell Lines Service, Heidelberg — the same catalog number as this item. The authors maintained A431 in RPMI-1640 with 10% FBS, whereas the supplier record specifies DMEM. Culture medium for this line is not standardized across the literature.
A431 and a melanoma line diverged at the level of individual respiratory-chain subunits
Wendt F, Wittig F, Rupprecht A, Ramer R, Langer P, Emmert S, Frank M, Hinz B. Cells 2023;12(19):2409. DOI · PMID 37830623
A431's roleThe cutaneous SCC half of a paired skin-cancer panel, run against A375 melanoma. This melanoma-plus-SCC pairing is the dominant convention across this cluster and is what allows lineage-specific effects to surface.
The compound was KD87, a thia-analogous indirubin N-glycoside. Beyond viability, the group profiled mitochondrial respiration on a Seahorse XFe24 analyser (oligomycin, FCCP, then antimycin A with rotenone) and examined mitochondrial ultrastructure.
- In both lines KD87 reduced viability, activated caspases-3 and -7, and inhibited colony formation.
- Concentration-dependent falls in basal and ATP-linked oxygen consumption rate and spare respiratory capacity, with cytochrome c release into the cytosol.
- A lineage-specific divergence: SDHB (complex II) fell in both A375 and A431, whereas NDUFB8 (complex I) fell only in A375. The two skin cancer lines were not interchangeable.
- Heme oxygenase-1 was upregulated; inhibiting it partially rescued the drop in metabolic activity — placing HO-1 upstream of part of the cytotoxic effect.
Reading the clusterThe complex I/complex II split only appears when a panel is used and subunits are measured individually. A companion reference is the LC-MS/MS bioanalytical method for this indirubin series — useful if you need a documented quantitation route alongside a cell readout.
Three further application areas appear in the citation record but rest on fewer studies and are not profiled here: drug delivery and formulation (3), enzyme regulation and metabolic phenotyping (3), and tumor microenvironment and photodynamic therapy (2). All are catalogued in the references on the product page.
Cross-cutting observation: redox signaling is an active variable in A431
Nrf2 and its downstream effectors surface independently in three unrelated corners of this literature: nuclear Nrf2 translocation in the plasma work, heme oxygenase-1 as a partial mediator of indirubin cytotoxicity, and the identification of carbonyl reductase 3 (CBR3) as an Nrf2 target gene in a panel that included A431 (Ebert et al., Biochemistry 2010;49(39):8499–511; DOI · PMID 20806931). Three separate groups, three unrelated questions, one recurring axis. The practical consequence: treat oxidative-stress signaling in A431 as an active variable rather than a quiet background — the line already sits near its antioxidant ceiling, so any redox-perturbing intervention may produce effects that are partly adaptive-capacity artefacts rather than the mechanism under test.
Provenance: catalog number 300112 is traceable into the literature
The same catalog number appears in published methods
This item's manufacturer number is 300112, supplied by Cytion. The Nature Communications study above records its A431 as “catalog no. 300112” obtained from Cell Lines Service, Heidelberg.
Cytion is a registered brand of CLS Cell Lines Service GmbH (Eppelheim, Germany), operating under the Cytion name since 23 November 2023. The legal entity, products and specifications are unchanged, and either the CLS or Cytion catalogue number may be cited academically.
For a methods section, the catalog number is a stable identifier linking this item to prior published work — a stronger provenance statement than a line name alone, since “A431” is distributed by several banks whose records differ.
A separate reference in this survey obtained A431 from ATCC, illustrating that the line reaches the literature through multiple banks. Confirm the citation format and current catalogue designation with the documentation shipped with your lot.
A431 cell line profile and culture conditions
| Attribute | Specification |
|---|---|
| Species | Human (Homo sapiens) |
| Donor | Female, 85 years, adult |
| Disease | Squamous cell carcinoma (epidermoid carcinoma) |
| Morphology | Epithelial-like, flat polygonal; grows in clusters |
| Growth properties | Adherent monolayer |
| Biosafety level | BSL-1 |
| Receptors | EGF-binding sites (high density) |
| p53 status | p53 protein positive. Published work identifies the allele as the R273H hotspot mutant — confirm against your lot documentation. |
| Mutational profile | BRAF wild type at codon 600 (V600E-negative); MYC amplification at 8q24 |
| Tumorigenicity | Yes, in immunosuppressed mice |
| QC | STR profiling and mycoplasma testing; batch CoA supplied |
Culture conditions as supplied
| Parameter | Condition |
|---|---|
| Culture medium | DMEM (4.5 g/L glucose, 4 mM L-glutamine, 3.7 g/L NaHCO3, 1.0 mM sodium pyruvate) |
| Supplement | 10% FBS |
| Dissociation | Accutase |
| Seeding density | 1 × 104 cells/cm2 → confluent within 4 days |
| Post-thaw plating | 5 × 104 cells/cm2; allow ≥24 h to recover |
| Fluid renewal | 2–3 times per week |
| Cryopreservation | Growth medium + FBS + 10% DMSO, or a commercial cryomedium |
Condensed from the supplier handling record. Published work uses other media for this line, including RPMI-1640; consult the product data sheet before first passage. Verify lot-specific data against the batch certificate of analysis supplied with your vial.
Before you commit: five things to settle at study-design stage
1. HaCaT is the de facto comparator, and it is not optional
Not one of the five featured studies used A431 on its own. Two pair it with HaCaT keratinocytes, a third with A375 melanoma, and the imaging study sets it against A549 and MLS xenografts. Every selectivity, potency-ratio, or malignant-versus-normal claim rests on that second line.
2. Treat A431 as a mutant-p53 model
The published allele is TP53 R273H, a DNA-contact hotspot mutant with gain-of-function activity shown in this line to drive invasion through BCAR1. Any experiment involving p53-dependent apoptosis, DNA-damage response, or chemosensitivity should account for that.
3. Genotype limits which comparisons are valid
A431 is BRAF wild type at codon 600 and carries MYC amplification at 8q24. In BRAF-inhibitor work it is a BRAF-wild-type reference, not a V600E model. In MYC-pathway work the pre-existing amplification is a baseline feature to control for, not a result.
4. High EGFR density calibrates; it does not prove selectivity — and the ligand response reverses
A positive result on A431 confirms that a targeting moiety engages EGFR. Because low EGF stimulates proliferation while high EGF inhibits growth and induces differentiation, a proliferation assay at a single ligand concentration can return a result whose sign depends on where that concentration falls. Bracket the range.
5. Anatomical origin is recorded inconsistently across cell banks
This record, following the supplier and ATCC, describes A431 as derived from the epidermis/skin of an 85-year-old female. ECACC (85090402) and its resellers describe the source tumor as an epidermal carcinoma of the vulva. Both trace to the same 1973 Giard series and the same line; the discrepancy is in how the primary site was recorded. If you present A431 as a vulvar SCC model, cite the repository record that supports that description.
Ordering the A431 cell line
| Product | SKU | Catalog no. | Size | Price |
|---|---|---|---|---|
| A431 cell | BHC11100124 | 300112 | 1 cryovial | $395.00 |
10% off · Cell Lines — Limited-Time Offer
A431 falls within the current cell lines promotion. Apply the code at checkout: CELL10
- One-time use only — applies to a single order, so it suits a first order or a bulk purchase rather than repeat resupply.
- Valid across the cell lines catalog (31,000+ lines, STR-profiled and mycoplasma-free, including primary cells, iPSCs, and disease models).
Limited-time offer. Confirm the code, current validity, and any exclusions on the promotions page or at checkout before ordering. Browse the full cell lines catalog.
Access agreements apply before shipment
This cell line is available in the United States and requires a completed agreement before an order can ship:
- Non-profit users — sign and submit the Non-Profit Supply Agreement to orders@biohippo.com.
- Commercial users — complete the CLEAR Form before ordering. Additional usage fees may apply depending on intended use.
Ships frozen on dry ice in cryotubes once the required agreement is complete; typical delivery is 2–3 business days. Each cryotube typically contains 3 × 106 cells. On receipt, inspect for damage and transfer immediately to below −130 °C.
A431 cell line: frequently asked questions
What is the A431 cell line used for?
A431 is a human epidermoid (squamous cell) carcinoma line most commonly used as (1) a high-EGFR model to calibrate EGFR-directed binders and probes, (2) a malignant keratinocyte counterpart to HaCaT in skin-cancer studies, (3) a reliably tumor-forming, highly angiogenic xenograft substrate, and (4) a cytotoxicity/natural-product screening line — usually paired with a second cell line.
What is the p53 status of A431 cells?
A431 is p53-protein positive, and published work identifies the allele as the TP53 R273H DNA-contact hotspot mutant with gain-of-function behaviour. Treat A431 as a mutant-p53 model, not a p53-competent one, and confirm the allele against your lot documentation.
What comparator cell line is used with A431?
HaCaT immortalized keratinocytes are the de facto non-malignant comparator. Other common counterparts include A375 melanoma (lineage-specific effects), A549/MLS (contrasting xenograft vasculature), and H1299 (p53-null reconstitution).
What culture medium do A431 cells need?
The supplier specifies DMEM (4.5 g/L glucose) with 10% FBS and Accutase for dissociation. Note that published studies also use RPMI-1640; medium for this line is not standardized across the literature, so follow the product data sheet for your lot.
Is A431 a squamous cell carcinoma line?
Yes — A431 is an epidermoid (squamous cell) carcinoma line. Some cell banks record the primary site as skin/epidermis and others as vulva; both trace to the same 1973 Giard series and the same line.
Featured references (verified against PubMed). The full 37-publication list is available on the product page and in the source application note.
Featured studies
- Cichocki M, et al. Effect of resveratrol and its methylthio-derivatives on EGFR and Stat3 activation in HaCaT and A431 cells. Mol Cell Biochem 2014;396(1–2):221–8. DOI · PMID 25063218Featured
- Guo AK, et al. Mutant TP53 interacts with BCAR1 to contribute to cancer cell invasion. Br J Cancer 2021;124(1):299–312. DOI · PMID 33144694Featured
- Manzhula K, et al. Nrf2/NF-κB signalling after plasma stimulation of malignant vs. non-malignant dermal cells. Int J Mol Sci 2024;25(20):10967. DOI · PMID 39456749Featured
- Opacic T, et al. Motion model ultrasound localization microscopy for tumor characterization. Nat Commun 2018;9:1527. DOI · PMID 29670096Featured
- Wendt F, et al. A thia-analogous indirubin N-glycoside disrupts mitochondrial function in melanoma and cSCC cells. Cells 2023;12(19):2409. DOI · PMID 37830623Featured
- Ebert B, et al. Regulation of human carbonyl reductase 3 (CBR3) by Nrf2 in cultured cancer cells. Biochemistry 2010;49(39):8499–511. DOI · PMID 20806931
For Research Use Only. Not for use in diagnostic or therapeutic procedures. A431 is supplied by Cytion, a registered brand of CLS Cell Lines Service GmbH; specifications, handling conditions, and documentation are those of the manufacturer. Published findings are summarized from the cited primary sources. Cell line characteristics, culture conditions, and pricing reflect the catalog record at the time of writing and are subject to change. Inclusion of a reference does not imply the authors used material supplied by BioHippo or Cytion, nor that they endorse this product.