sh at every media change. Control for in vitro differentiation assays was culture media alone.
Wild-type (AB; Zebrafish International Resource Center, Naquotinib (mesylate) Eugene, OR, USA) zebrafish were bred and embryos were raised following procedures previously described [26]. Adult zebrafish were housed in 10 liter (L) aquaria at a density of ~5 fish per 1L with a 14 h/10 h light/ dark cycle. Fish were fed Zeigler Adult Zebrafish Diet (Pentair Aquatic Eco-Systems) twice daily and recordings of water temperature (~27.5), pH (7.5), conductivity (800 S) were collected daily. Single embryos were cultured in individual wells of multiwall plates to permit individual dosing and phenotyping. To assess vitality and growth following extract exposures: survival, hatching from chorion and pigment formation (Full, partial or none) were assessed every 24 h. At approximately 72 hours post exposure (hpe), incidence and severity of heart malformation was scored. Heart rate was determined by counting ventricular contractions over a period of one minute from randomly selected zebrafish larvae at 27. For qRT-PCR, zebrafish embryos (cleavage stage) were exposed to either control, e-cigarette or tobacco extracts at 13.7 M and embryos were collected at 24 hpe for RNA isolation as described below. Bright field images were obtained with a Nikon SMZ1000 microscope using a Canon Rebel T3i camera. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Washington, Seattle. All assays consist of a minimum of three independent breeding trials and data were collected in a blinded fashion.
Undifferentiated RUES2 hESCs (Female line, Rockefeller University, 10205015 NIH registry number 0013) were plated at 
1.6×105 cells/cm2 on Matrigel (BD) coated plates and maintained in an undifferentiated state with mouse embryonic fibroblast (MEF) conditioned media containing 5 ng/mL hbFGF (Peprotech, 100-18B). Directed differentiations using a monolayer platform were performed based on previous reports [27] with a modified protocol. Undifferentiated hESCs were plated as single cells as described previously and upon reaching appropriate confluency, treated with the Wnt/-catenin agonist CHIR-99021 (1 M, Cayman chemical, 13122) for 24 hours. Cells were then exposed to Activin A (R&D SYSTEMS, 338-AC-050) (100 ng/ mL) in RPMI/B27 medium (day 0). After 17 hours, media was changed to RPMI/B27 medium containing BMP4 (R&D SYSTEMS, 314-BP-050) (5 ng/mL) and CHIR-99021 (1 M, Cayman chemical,13122). On day 3, media was changed to RPMI/B27 medium containing the Wnt/catenin antagonist XAV-939 (1 M; Tocris, 3748). Media was then changed on day 5 to RPMI/ B27 medium. From day 0 to day 5, the B27 supplement utilized did not contain insulin (Invitrogen, 0050129SA). From day 74 a B27 supplement with insulin was used (Invitrogen, 17504044). For assays assessing the onset and rate of beating, cultures were analyzed independently during differentiation, with each well counted as n = 1.
For quantitative RT-PCR, total RNA was isolated using the RNeasy Miniprep kit (Qiagen). RNA quality and amount was determined using a Nanodrop spectrophotometer. First-strand cDNA was synthesized was performed using the Superscript III enzyme kit (Invitrogen). Quantitative RT-PCR was performed using Sensimix SYBR PCR kit (Bioline) on a 7900HT FastReal-Time PCR System (Applied Biosystems). For in vitro assays, the copy number for each transcript is expressed relative to that o