SUPPLEMENTARY METHOD
Cell proliferation assay (BrdU incorporation)
Cells (5 × 103 cells) were seeded in 96-well plates in 100 μl of cell suspension in each well. After
24 h, they were treated with or without IL-33 for 24 h, labeled with 10 µL/well BrdU labeling
solution, and then incubated for 4 h at 37°C in a 5% CO2 atmosphere. Cell proliferation was
estimated by measuring the absorbance at 370 nm.
Measurement of cytokines
The cells were plated in 96 well plates at 5 ×103 cells/well overnight and treated with IL-33 for
an additional 24 h. Culture media was harvested for measurement of IL-6, MCP-1, and TNF-α
concentration. The ELISA was performed according to the manufacturer instructions (R&D
Systems, Minneapolis, MN, USA). The absorbance of the reaction solution was measured at 450
nm on a Microplate Autoreader ELISA (Bio-Tek Instruments Inc., Winooski, VT, USA).
Immunoblot analysis
The cells were disrupted in radioimmunoprecipitation assay (RIPA) lysis buffer. The proteins
were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred onto PVDF. For detecting chemiluminescence, a LAS 4000 imaging system (GE
Healthcare Biosciences, Pittsburgh, PA, USA) was used.
Anchorage-independent cellular transformation assay
Briefly, 8 × 103 cells were exposed to different doses of IL-33 in 1 mL of 0.3% basal medium
Eagle’s (BME) agar containing 10% FBS, 2 mM L-glutamine, and 25 µg/mL gentamicin. The
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cultures were maintained at 37°C in a 5% CO2 incubator for 14 days, and scores of the cell
colonies were determined using an Axiovert 200 M fluorescence microscope and the Axio
Vision software (Carl Zeiss, Inc., Thornwood, NY, USA).
Reporter gene assays
To detect firefly luciferase activity, the reporter gene assay was performed using lysates from cfos-, c-jun-, AP-1-, or stat3-luc-transfected JB6 Cl41 cells. The reporter gene vector pRL-TKluciferase plasmid (Promega) was co-transfected into each cell line and the Renilla luciferase
activity generated by this vector was used to normalize the results for transfection efficiency.
Cell lysates were mixed with luciferase assay II reagent, and firefly luciferase light emission was
measured using the GloMax®-Multi Detection System (Promega). Subsequently, Renilla
luciferase substrate was added to normalize the firefly luciferase data. The c-fos-luc promoter
(pFos-WT GL3) and c-jun-luc promoter (JC6GL3) constructs were kindly provided by Dr Ron
Prywes (Columbia University, New York, NY). The AP-1 and stat3 luciferase reporter plasmid
(−73/+63 collagenase-luciferase) were kindly provided by Dr Dong Zigang (Hormel Institute,
University of Minnesota, Austin, MN) and Dr K-Y Lee (Chonnam National University, South
Korea).
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FIGURE LEGENDS
Supplementary Figure S1
(a − c) Production of IL-6, MCP-1, and TNF-α in MCF-7 and MDA-MB-231 cells in response to
IL-33. The cells were treated with IL-33 for 24 h. IL-6, MCP-1, and TNF-α levels in the culture
supernatant were measured by ELISA, as indicated in Materials and Methods. Data are shown as
means ± SEM of triplicate samples from one experiment representative of three independent
experiments performed. **, P < 0.01, ***, P < 0.001.
Supplementary Figure S2
(a and b) Cell proliferation was estimated using a BrdU assay. Left panels, MCF7 cells (a) or
MDA-MB231 cells (b) were seeded, maintained for 24 h, and then treated or not treated with 50
ng/ml IL-33 for 24 h; right panels, MCF7 cells (a) or MDA-MB231 cells (b) were seeded,
maintained for 24 h Then the cells were stimulated with the supernatants from IL-33-treated
MCF7 cells (IL33 MCF7 SN) and MDA-MB231 cells (IL33 MDA-MB231 SN) for 24 h, which
were treated with/without 25 ng/ml or 50 ng/ml IL33 for 24 h, respectively. Data are represented
as mean ± SEM from one experiment representative of three independent experiments performed
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