Chemical mixtures isolated from house dust disrupt thyroid receptor β (TRβ) signaling

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1 SUPPORTING INFORMATION Chemical mixtures isolated from house dust disrupt thyroid receptor β (TRβ) signaling Erin M. Kollitz, Christopher D. Kassotis, Kate Hoffman, P. Lee Ferguson, Julie Ann Sosa, Heather M. Stapleton * Nicholas School of the Environment, Duke University, Box 90328, Durham, North Carolina 27708, United States Department of Surgery, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, CA 94117, United States Number of pages: 7 Number of tables: 3 Number of figures: 1 CONTENTS PAGE # Table S1: Chemicals used in the GeneBLAzer assay... S2 Table S2: Cell culture reagents and plastics used in the GeneBLAzer assay... S3 Table S3: Health metrics summary... S4 SI Methods: Frozen cell stocks... S5 SI Methods: Cell viability assay... S6 Figure S1: T3 dose-response curve... S7 S1

2 Table S1 Table S1. Chemicals used in the GeneBLAzer assay. Chemical Name Abbreviation CAS # Catalog # Purity Supplier Location 2,2',4,4'-tetrabromodiphenyl ether BDE BDE-047N 99% AccuStandard New Haven, CT 2,2',4,4',5-pentabromodiphenyl ether BDE BDE-099N 97% AccuStandard New Haven, CT 2,2',4,4',6-pentabromodiphenyl ether BDE BDE-100N 99% AccuStandard New Haven, CT 2,2',4,4',5,5'-hexabromodiphenyl ether BDE BDE-153N 99% AccuStandard New Haven, CT 2,2',4,4',5,6'-hexabromodiphenyl ether BDE BDE-154N 99% AccuStandard New Haven, CT 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether BDE BDE-209N 98% AccuStandard New Haven, CT 2-ethylhexyl-tetrabromobenzoate EH-TBB FRS-041N 95% AccuStandard New Haven, CT Bis(2-ethylhexyl)-tetrabromophthalate BEH-TEBP FRS-040N 99% AccuStandard New Haven, CT Tris(2-chloroethyl) phosphate TCEP % Sigma-Aldrich St. Louis, MO Tris(1,3-dichloroisopropyl) phosphate TDCIPP % Sigma-Aldrich St. Louis, MO Tris (1-chloro-2-propyl) phosphate TCIPP % Sigma-Aldrich St. Louis, MO Triphenyl phosphate TPHP % Sigma-Aldrich St. Louis, MO L-3,3',5-Triiodothyronine, Free Acid T T0453 >98% TCI America Portland, OR Resazurin Sodium Salt Rez R % Sigma-Aldrich St. Louis, MO S2

3 Table S2 Table S2. Cell culture reagents and plastics used in the GeneBLAzer assay. Name Catalog # Supplier Location DMEM, high glucose Invitrogen Carlsbad, CA DMEM, high glucose, phenol red free Invitrogen Carlsbad, CA Recovery Cell Culture Freezing Medium Invitrogen Carlsbad, CA Fetal Bovine Serum, charcoal-stripped Invitrogen Carlsbad, CA Fetal Bovine Serum, Dialyzed Invitrogen Carlsbad, CA Non-essential Amino Acids (NEAA, 10 mm) Invitrogen Carlsbad, CA Sodium Pyruvate (100 mm) Invitrogen Carlsbad, CA GlutaMAX (200 mm) Invitrogen Carlsbad, CA HEPES (1M) Invitrogen Carlsbad, CA Pen/Strep (10,000 U/mL) Invitrogen Carlsbad, CA Hygromycin B (50 mg/ml) Invitrogen Carlsbad, CA Zeocin Selection Reagent (100 mg/ml) R Invitrogen Carlsbad, CA Trypsin/EDTA (0.05%) Invitrogen Carlsbad, CA Dimethyl Sulfoxide (DMSO) D2650 Sigma-Aldrich St. Louis, MO LiveBLAzer -FRET B/G Loading Kit K1095 Invitrogen Carlsbad, CA 384-well plate, TC-treated, black/clear 3764 Corning Inc. Corning, NY T-75 vented tissue culture flasks U Corning Inc. Corning, NY Cryogenic Vials, 2 ml, sterile Fisher Scientific Hampton, NH S3

4 Table S3 Table S3. Health metrics summary. Health Metric Body mass index (BMI) Serum cholesterol Sample Size Mean Median Range Overall Males Females Overall Males Females Serum triglycerides Overall Males Females Thyroid stimulating hormone (TSH) Overall Males Females Free thyroxine (FT4) Overall Males Females Free triiodothyronine (FT3) Overall Males Females S4

5 Supporting Materials and Methods Frozen Cell Stocks. TRβ-UAS-bla HEK293T cells (catalog #K1686, Lot # ) were obtained from Invitrogen (Carlsbad, CA). Information regarding cell culture reagents and plastics used in this assay, along with catalog numbers and manufacturers, can be found in Table S2. Cells were thawed following the manufacturer s protocol [59], and cultured for three passages in high glucose Dulbecco s Modified Eagle Medium (DMEM) supplemented with 10% dialyzed fetal bovine serum (FBS), 4 mm GlutaMAX, 25 mm HEPES, 0.1 mm non-essential amino acids, 1 mm sodium pyruvate, 100 U/mL penicillin and streptomycin, 80 µg/ml hygromycin-b, and 100 µg/ml Zeocin. Cells were passaged at 80% confluency following the manufacturer s protocol using 0.05% Trypsin with EDTA. At the third passage, cells were harvested and re-suspended at a density of 4.6 x 10 6 cells/ml in Recovery Cell Culture Freezing Medium. The cell suspension was divided into 1 ml single-use aliquots and slowly cooled (-1 C/min) in an insulated slow freezing container stored at -80 C overnight. Frozen cell stocks were stored in liquid nitrogen until use. S5

6 Supporting Materials and Methods Cell Viability Assay. Cell viability was assessed simultaneously with each GeneBLAzer plate using a resazurin reduction assay [61]. Stock solutions (0.15 mg/ml) were prepared by dissolving resazurin sodium salt (Sigma-Aldrich Corp., St. Louis, MO) in cell culture grade Dulbecco s phosphate buffered saline (ph 7.4, Invitrogen, Carlsbad, CA). Stocks were filter-sterilized and stored at -20 C in single-use aliquots. To assess cell viability, 8 µl of the resazurin stock solution was added to each well (1x = mg/ml) and incubated for two hours at 37 C in a humidified CO 2 incubator. Fluorescence was measured at 560/590 nm. Fluorescent background was first subtracted from each well, and the % viability was determined with the following formula: % Viability= Fluorescence Fluorescence 100 Cell viability loss of greater than 15% was used as the cut-off for distinguishing cytotoxicity from antagonism. Cytotoxic wells and doses were excluded from further analysis. S6

7 Figure S % Activation Log T3 (nm) Figure S1. T3-TRβ dose-response curve. TRβ-UAS-bla HEK293T cells were plated into 384-well plates (~10,000 cells/well) and treated with nm of T3 for 18 hours. TRβ activity was assessed by measuring β-lactamase activity as described in the Materials and Methods. The plotted data represents the average TRβ activation as a percent relative to the maximum activation control and is the average ± standard deviation of three separate experiments (4 wells per dose in each experiment). The calculated EC 50 for T3 was nm (95% confidence interval: nm). S7