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Method Article

Establishment of a STAT6 Reporter Assay for Screening Environmental Toxicants Affecting Allergic Airway Inflammation

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DOI:

10.3791/71694

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August 25th, 2026

 ,  ,  ,  , 
1Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University

In This Article

Summary

This protocol describes the generation and validation of a stable STAT6 luciferase reporter assay in human airway epithelial cells. The method enables efficient screening of environmental toxicants and air pollution components that modulate STAT6 activity under controlled in vitro conditions.

Abstract

Air pollution–associated allergic airway inflammation is an increasing public health concern. Interleukin‑4 (IL‑4) and interleukin‑13 (IL‑13), which activate the Signal Transducer and Activator of Transcription 6 (STAT6) pathway, a central mediator of allergic airway inflammation, may modulate the respiratory toxicities of pollutants. The present study describes the generation and validation of a stable STAT6 luciferase reporter assay in human airway epithelial cells for evaluating environmental toxicants that modulate STAT6 signaling. Human bronchial epithelial 16HBE14o− cells were transduced with a STAT6-responsive luciferase reporter using a lentiviral vector, followed by optimization of puromycin selection and multiplicity of infection, and monoclonal isolation by limiting dilution. A stable clone with strong and reproducible induction across serial passages was selected. Reporter responsiveness was validated by IL-4/IL-13 stimulation, and STAT6 dependence was confirmed using selective STAT6, STAT5, and STAT3 inhibitors. Assay performance was quantified by Z′-factor analysis, which indicated reproducible signal separation. Furthermore, the assay was applied to individual air-pollution constituents, and benzo[b]fluoranthene and particulate matter significantly increased STAT6 reporter activity. This method provides a scalable approach for measuring STAT6 activity in airway epithelial cells and for prioritizing environmental toxicants that modulate allergic airway signaling.

Introduction

Air pollution represents one of the most pressing global public health challenges, with profound health effects and socioeconomic impacts. It is currently ranked as the second leading risk factor for mortality, accounting for 8.1 million deaths annually1,2,3. Among airborne pollutants, fine particulate matter with diameters of less than 2.5 micrometers (PM2.5) poses the greatest health risk due to its ability to penetrate deep into the respiratory tract4,5. Elevated PM2.5 exposure has been consistently linked to impaired lung function and increased rates of hospitalization for respiratory diseases6. The prevalence of allergic inflammatory diseases, including asthma, allergic rhinitis, and atopic dermatitis, has been increasing worldwide and is disproportionately elevated in the highly polluted megacities4.

Allergic airway diseases such as atopic asthma are primarily mediated by a type 2 inflammatory response characterized by elevated levels of the cytokines interleukin‑4 (IL‑4) and interleukin‑13 (IL‑13) and activation of the Signal Transducer and Activator of Transcription 6 (STAT6) signaling pathway7. In airway epithelial cells, IL-4- and IL-13-driven phosphorylation of STAT6 promotes mucus hypersecretion and epithelial barrier disruption, identifying STAT6 as a central mediator of allergic airway pathology and a relevant therapeutic target8,9. Experimental evidence from murine models indicates that exposure to PM2.5 and sulfur dioxide augments STAT6 signaling and exacerbates allergic airway inflammation10,11. However, whether individual air pollution constituents directly modulate STAT6 transcriptional activity in human airway epithelial cells has not been systematically evaluated.

Air pollution comprises a chemically complex mixture of constituents, including particulate matter, polycyclic aromatic hydrocarbons (PAHs), heavy metals, and inorganic salts, whose individual contributions to airway STAT6 activation remain poorly defined12,13. Current methodologies for measuring STAT6 activity in airway epithelial cells, such as endpoint phosphorylation assays or transient transfection-based reporter systems, are not amenable to large-scale chemical screening. A stable, cell-based STAT6 reporter platform in human airway epithelial cells would therefore provide a scalable tool to identify pollution-derived compounds that modulate allergic airway signaling. This protocol describes the development and validation of a stable STAT6 luciferase reporter assay in 16HBE14o− airway epithelial cells, enabling quantitative evaluation of STAT6 activation for systematic screening of environmental samples and individual chemical toxicants.

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Protocol

Human-subject ethics approval is not applicable because this study used the established 16HBE14o− human bronchial epithelial cell line.

1. Generation of 16HBE14o− cells stably expressing a STAT6 activity reporter

NOTE: Figure 1 provides an overview of the workflow.

  1. Cell culture
    NOTE: Perform all procedures using aseptic technique in a Class II biosafety cabinet. Confirm that the cells are free of mycoplasma contamination before use.
    1. Maintain the 16HBE14o− cells in minimum essential medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Incubate the cells at 37 °C in a humidified atmosphere containing 5% CO2, and replace the culture medium every other day.
    2. Allow the cells to reach approximately 80% confluence. Aspirate the culture medium and rinse the cell monolayer once with phosphate-buffered saline.
    3. Add sufficient phosphate-buffered saline to cover the cell monolayer and incubate the cells for 20 min at 37 °C. Aspirate the phosphate-buffered saline completely.
      NOTE: Use the 20 min phosphate-buffered saline incubation to facilitate disruption of the tight intercellular junctions formed by this cell line. This duration represents an in-house optimized modification.
    4. Add sufficient 0.25% trypsin–ethylenediaminetetraacetic acid solution to cover the cell monolayer. Incubate the cells for 10 min at 37 °C.
      NOTE: Monitor cell detachment using an inverted light microscope. Gently tap the culture vessel when necessary to facilitate detachment.
    5. Add approximately five volumes of complete culture medium relative to the volume of trypsin–ethylenediaminetetraacetic acid solution to terminate enzymatic activity. Pipette the suspension gently to obtain a single-cell suspension.
    6. Transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge the suspension at 300 × g for 5 min at 25 °C.
    7. Aspirate the supernatant carefully and resuspend the cell pellet in fresh culture medium. Gently triturate the suspension and confirm the presence of a single-cell suspension under an inverted light microscope.
    8. Count the cells using a hemocytometer and adjust the cell concentration to the density required for the subsequent experiment. Use a routine split ratio of 1:8.
      NOTE: Refer to Supplementary Table 1 and Supplementary Table 2 for the growth areas of the culture vessels and the corresponding volumes of phosphate-buffered saline and trypsin–ethylenediaminetetraacetic acid solution.
  2. Determination of puromycin-induced cytotoxicity
    ​NOTE: Select the antibiotic according to the resistance gene encoded by the lentiviral construct.
    1. Seed the 16HBE14o− cells at 2 × 104 cells per well in a 96-well culture plate containing 100 µL of complete culture medium per well. Incubate the cells overnight.
    2. Replace the culture medium with fresh medium containing puromycin at final concentrations of 0.5, 1, 2, or 4 µg/mL. Prepare separate plates for the 24-, 48-, and 72-h time points.
    3. Add 5 µL of cell-counting reagent to each well. Incubate the plate for 1 h at 37 °C.
      NOTE: Avoid introducing air bubbles into the wells, as they may interfere with absorbance measurements.
    4. Measure the absorbance at 450 nm using 650 nm as the reference wavelength. Shake the plate for 15 s immediately before recording the absorbance in endpoint mode.
    5. Calculate cell viability14 using Equation 1.
      Cell viability equation, OD450-650 for samples, control, blank; formula for bioassay analysis.
      where:
      Absorbance equation OD450-650,subtraction method, used in spectrophotometry, formula.
      Optical density formula (OD450-650) for absorption analysis in spectroscopy experiments.
      Optical density equation, OD450-650blank=OD450blank-OD650blank, for spectroscopic data analysis.
      NOTE: Define the control as untreated cells and the blank as cell-free medium containing the assay reagent. Select the lowest puromycin concentration that completely eliminates the non-transduced cells within 48–72 h.
  3. Determination of the optimal multiplicity of infection
    CAUTION: Handle lentiviral particles under Biosafety Level 2 conditions in a registered Biosafety Level 2 facility and in accordance with institutional biosafety regulations. Perform all virus manipulations in a Class II biosafety cabinet. Cover virus-contaminated consumables and spills with 10% bleach for at least 1 h before disposal as biohazardous waste.
    ​NOTE: Optimize the multiplicity of infection because it directly affects transduction efficiency.
    1. Calculate the required volume of lentiviral stock15 using Equation 2.
      MOI calculation formula, ratio of virus particles to target cells, molecular biology equation.
      where:
      Functional virus titer formula, T_virus = TU/mL, equation for virology research data analysis.
      Virus volume equation, V_virus=virus volume in mL, formula, virology study, calculation method
      Static equilibrium, equation: N_cells = number of target cells, diagram for educational research use.
      NOTE: The functional titer of the STAT6 luciferase reporter lentiviral stock used in this protocol was 5 × 107 transducing units/mL. For 2 × 104 target cells, an MOI of 20 corresponds to 8 µL of lentiviral stock per well. The virus volume should be recalculated for virus preparations with different titers.
    2. Seed the 16HBE14o− cells at 2 × 104 cells per well in a 96-well white clear-bottom plate containing 100 µL of complete culture medium per well. Incubate the cells overnight until they reach approximately 80% confluence.
    3. Replace the culture medium with fresh medium containing 5 µg/mL polybrene and STAT6 luciferase reporter lentivirus at MOIs of 1, 5, 10, and 20. Include a non-transduced mock-control well containing no lentivirus.
      NOTE: Calculate the lentiviral volume required for each MOI from the functional titer of the stock.
    4. Incubate the cells with the lentiviral particles for 48 h. Replace the transduction medium with fresh culture medium and incubate the cells for an additional 24 h.
    5. Replace the culture medium with fresh medium containing interleukin-4 and interleukin-13 at 10 ng/mL each. Incubate the cells for 24 h.
    6. Add 25 µL of luciferase assay reagent to each well16. Protect the plate from light and incubate it for 20 min at room temperature.
    7. Shake the plate linearly for 20 s. Measure luminescence in endpoint mode using a full-plate read and a gain setting of 120.
      CAUTION: Record luminescence within 1 h after adding the luciferase assay reagent to capture the maximal signal. Follow the reagent manufacturer’s safety and handling instructions.
      NOTE: Use the same multimode microplate reader and acquisition settings for all absorbance and luminescence measurements.
  4. Selection of transduced cells
    1. Seed the 16HBE14o− cells at 2 × 105 cells/mL in a 24-well culture plate. Incubate the cells overnight.
    2. Replace the culture medium with fresh medium containing the STAT6 luciferase reporter lentivirus at an MOI of 20 and 5 µg/mL polybrene. Incubate the cells for 48 h.
    3. Replace the transduction medium with fresh culture medium. Incubate the cells for an additional 24 h.
    4. Aspirate the culture medium and add fresh medium containing 1 µg/mL puromycin. Incubate the cells for 5 days, replacing the selection medium every 2 days.
    5. Continue puromycin selection until all cells in the non-transduced mock-control well are eliminated.
      ​NOTE: Allow at least 24 h after transduction before applying antibiotic selection to permit expression of the resistance gene.
  5. Isolation of single-cell clones by limiting dilution
    1. Detach and count the puromycin-selected STAT6 reporter cells. Prepare a single-cell suspension at 5 cells/mL by serial dilution.
      ​NOTE: Prevent cell clumping during dilution. Pass the suspension through a cell strainer when necessary.
    2. Dispense 200 µL of the cell suspension into each well of a 96-well culture plate to obtain an average density of approximately one cell per well.
    3. Examine each well using an inverted microscope within 24 h and identify wells containing a single cell. Exclude wells containing more than one cell or multiple spatially separated cell foci.
    4. Monitor the selected wells during the first week to confirm clonal growth. Replace the culture medium every 3 days.
    5. Expand each clone in the original 96-well plate for approximately 20 days or until the cells reach confluence. Transfer each clone sequentially to a 6-well plate and then to a T-25 culture flask.
      NOTE: Culture the cells in the 6-well plate for approximately 6 days before transferring them to the T-25 flask, or transfer them when they reach the appropriate confluence.
      ​PAUSE POINT: Continue expanding the clones or cryopreserve them at 1 × 106 cells per vial in 1 mL of cryopreservation medium containing 90% fetal bovine serum and 10% dimethyl sulfoxide.
    6. Seed each expanded clone at 2 × 105 cells/mL in a 96-well white clear-bottom plate. Incubate the cells overnight.
    7. Replace the culture medium with fresh medium containing IL-4 and IL-13 at 10 ng/mL each. Incubate the cells for 24 h.
    8. Measure luciferase activity as described in steps 1.3.6–1.3.7. Compare the induced reporter activity among the individual clones.
    9. Repeat the luciferase assay over increasing cell-passage numbers. Select clones that maintain consistent cytokine-induced reporter activity across passages.
      NOTE: Clone 3 was selected for its low basal luminescence and ability to maintain a consistent cytokine-induced activation signal across serial passages.

2. Establishment and validation of the STAT6 activity reporter assay

NOTE: Figure 2 provides an overview of the workflow.

  1. Validation of the STAT6 activity reporter assay
    1. Seed the STAT6 reporter cells at 2 × 105 cells/mL into a 96-well white clear-bottom plate using 100 µL of complete culture medium per well. Incubate the cells overnight.
    2. Replace the culture medium with fresh medium containing IL-4 and IL-13 at final concentrations of 10 ng/mL each. Add the selective inhibitors simultaneously as follows: 100 nM AS1517499 (STAT6 inhibitor), 2.5 µM AC-4-130 (STAT5 inhibitor), or 2.5 µM Stattic (STAT3 inhibitor).
    3. Include a vehicle control containing 0.1% dimethyl sulfoxide (DMSO). Incubate the cells for 24 h.
    4. Determine cell viability using the cell-counting assay as described in steps 1.2.3–1.2.5.
    5. Measure luciferase activity as described in steps 1.3.6–1.3.7.
    6. Normalize the luminescence signal of each well to its corresponding cell viability. Express the normalized values relative to the cytokine-treated control group.
      NOTE: Reduced cell viability may decrease the luciferase signal independently of STAT6 activity. Interpret reporter activity only after confirming acceptable cell viability.
  2. Evaluation of assay robustness
    1. Evaluate assay robustness by calculating the Z′-factor using 21 replicate wells for both the positive- and negative-control groups.
    2. Calculate the Z′-factor using Equation 3.
      Static equilibrium equation, Z-factor formula, mathematical expression for assay quality control.
      where:
      Static equilibrium equation, σₚ denotes standard deviation in control, educational formula illustration.
      Standard deviation equation, σn, used in statistical analysis for negative control accuracy.
      Equation μ<sub>p</sub>=mean signal of the positive control; statistical data analysis concept.
      Static equilibrium, equation μn= mean signal of the negative control, educational analysis.
      NOTE: A Z′-factor between 0.5 and 1.0 indicates that the assay is suitable for high-throughput screening.

3. Application of the STAT6 reporter assay for screening environmental toxicants

CAUTION: Handle polycyclic aromatic hydrocarbons and heavy-metal salts as toxic and potentially carcinogenic materials. Use appropriate personal protective equipment, perform all manipulations in a certified chemical fume hood, and dispose of contaminated materials in accordance with institutional hazardous waste procedures.

  1. Environmental toxicant screening
    1. Seed the STAT6 reporter cells at 2 × 105 cells/mL into a 96-well white clear-bottom plate using 100 µL of complete culture medium per well. Incubate the cells overnight.
    2. Replace the culture medium with fresh medium containing each test compound at the desired final concentration, which is 10 µM for this protocol, and treat the cells in the absence of IL-4 and IL-13.
    3. Include a negative-control group containing 0.1% dimethyl sulfoxide and a positive-control group containing IL-4 and IL-13 at 10 ng/mL each. Incubate the cells for 24 h.
    4. Determine cell viability using the cell-counting assay as described in steps 1.2.3–1.2.5.
    5. Measure luciferase activity as described in steps 1.3.6–1.3.7.
    6. Normalize the luminescence signal from each well to the corresponding cell-viability value. Express the normalized result relative to the negative-control group.
    7. Exclude compounds that reduce cell viability below 80% from the interpretation of STAT6 reporter activity.
      NOTE: Treat reporter signals obtained under cytotoxic conditions as unreliable because reduced cell viability may alter luminescence independently of STAT6 pathway activity.

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Results

To determine the optimal puromycin concentration for stable selection, parental 16HBE14o− cells were treated with increasing concentrations of puromycin (0.5–4 µg/mL), and cell viability was assessed after 24, 48, and 72 h using a cell viability assay. Puromycin reduced cell viability in a dose- and time-dependent manner, with all concentrations decreasing viability to below 20% after 48–72 h (Figure 3). A concentration of 1 µg/mL was the lowest concentration that completely elimi...

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Discussion

IL-4/IL-13–JAK–STAT6 signaling is not only an important therapeutic target but also a key driver of the pathogenesis of allergic diseases, including asthma and allergic rhinitis22. This protocol describes a simple, stable cell line-based reporter assay for identifying modulators of STAT6 activity. The assay can be applied to evaluate the STAT6-modulatory effects of a broad range of environmental compounds and is readily adaptable to high-throughput screening, as demonstrated in the pro...

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Disclosures

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the National Research Council of Thailand (NRCT) (Grant No. N42A680388), and this research project is supported by the Mahidol University (Strategic Research Fund: 2025) (Grant No. 35). Figures 1 and 2 were created using BioRender.com (Pothipan, P. (2026). https://BioRender.com/h44gy3h).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.25% Trypsin-EDTAGibco25200072Cell dissociation reagent
15 mL conical centrifuge tubeCorningCLS430791Sterile polypropylene centrifuge tube
16HBE14o- human bronchial epithelial cell lineGift from Dr. Nawiya Huipao (Prince of Songkla University, Thailand) and Prof. Dr. Dieter C. Gruenert (University of California San Francisco, USA)RRID:CVCL_0112Human bronchial epithelial cell line (passages 35–40); confirmed mycoplasma-free and retains differentiated airway epithelial morphology and barrier properties. Reference: Cozens AL, et al. Am J Respir Cell Mol Biol. 1994;10(1):38–47.
24-well clear flat-bottom tissue culture-treated plateCorning3527Cell culture plate
6-well clear flat-bottom tissue culture-treated plateCorning3516Cell culture plate
96-well clear flat-bottom polystyrene tissue culture-treated microplateCorning3599Cell viability assay plate
96-well white clear-bottom polystyrene tissue culture-treated microplateCorning3610Luminescence assay plate
AC-4-130 (STAT5 inhibitor)MedChemExpressHY-124500Selective STAT5 inhibitor
AS1517499 (STAT6 inhibitor)MedChemExpressHY-100614Selective STAT6 inhibitor
Benzo[b]fluorantheneAccuStandardH-128NPolycyclic aromatic hydrocarbon
Biological safety cabinet, Class IINuaireNU-543-400EFor aseptic cell culture procedures
Cadmium chloride (CdCl2)Sigma202908Heavy metal compound
Cell counting kit-8 (CCK-8)MedChemExpressHY-K0301Cell viability assay
Cell culture flask, 25 cm² (T-25)Corning430639Tissue culture flask
CO2 incubatorNuaireNU-5710EMaintained at 37 °C, 5% CO2, humidified atmosphere
Dimethyl sulfoxide (DMSO)SigmaD2650Solvent (0.1% final concentration) and cryoprotectant (10%)
Fetal bovine serum (FBS)SigmaF7524Medium supplement (10%) and cryopreservation medium (10%)
HemocytometerBOECOBOE14Manual cell counting
Inverted phase-contrast light microscopeZEISSPrimovertCell morphology observation
Luciferase assay system (ONE-Step)BPS Bioscience60690Luciferase reporter assay
Mercury chloride (HgCl2)Sigma215465Heavy metal compound
Microplate readerBioTekSynergy Neo2Multimode microplate reader/luminometer controlled by Gen5 software (v3.08.01). Absorbance measured at 450 nm with 650 nm reference after 15–20 s linear shaking. Luminescence measured in endpoint mode using detector gain of 120 following 20 s linear shaking.
Minimum Essential Medium Eagle (MEM)Gibco11095-080Basal culture medium
Nickel sulfate (NiSO4)Tokyo Chemical IndustryN1203Heavy metal compound
Particulate matter (PM)National Institute of Standards and TechnologySRM 2975Standard reference material for particulate matter
Penicillin-streptomycin (100×)Gibco15140122Antibiotic supplement (1% final concentration)
Phosphate-buffered saline (PBS)Gibco18912014Washing buffer
PolybreneSigmaTR-1003-GLentiviral transduction enhancer
PuromycinMedChemExpressHY-B1743AAntibiotic for stable cell selection
Recombinant human IL-13Thermo Fisher ScientificA42526Cytokine for stimulation
Recombinant human IL-4Thermo Fisher ScientificA42602Cytokine for stimulation
STAT6 luciferase reporter lentivirusBPS Bioscience78799Lentiviral reporter construct; stock titer 5 × 107 TU/mL
Stattic (STAT3 inhibitor)MedChemExpressHY-13818Selective STAT3 inhibitor

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