Cell counting assays MDA MB 468, MDA MB 468shAhR, MCF7, MDA MB 231, Cal51, and Cal51shAhR selleck were seeded at 20,000 cells/ well and 15,000 cells/well, each in triplicate 12 well tissue culture plates in DMEM 10% FBS at 37 C and 5% CO2. AhR knockdown cells were pretreated with vehicle or Inhibitors,Modulators,Libraries 750 ng/mL Dox for seven days prior to seeding in 12 well tissue culture Inhibitors,Modulators,Libraries plates to achieve knockdown of AhR. During AF treatment, vehicle/Dox treatments were continued. All cell lines tested were treated with AF for seven days prior to analysis. Approxi mate GI50 value, which is the concentration of compound that inhibits cell growth by 50% compared to control, was calculated using GraphPad Prism Software and a three parameter log versus inhibition nonlinear regression. GI50 values are expressed as the 95% confidence interval.

Gene expression analysis MDA MB 468, MDA MB 468shAhR, Cal51, and Cal51 shAhR cells were cultured in phenol red free DMEM 10% charcoal Inhibitors,Modulators,Libraries stripped FBS at 37 C and 5% CO2 for three days prior to experiment to remove residual estrogens. Triplicate 80% confluent six cm tissue culture dishes of MDA MB 468 and Cal51 were treated with 0. 1% DMSO, 1 uM AF, or 1 uM BNF for six hours. MDA MB 468shAhR and Cal51shAhR were pretreated with vehicle or 750 ng/mL Dox for seven days prior to seeding onto triplicate six cm tissue culture dishes, and then treated with 0. 1% DMSO, 1 uM AF, or 1 uM BNF for six hours in the presence or absence of 750 ng/mL Dox. Total RNA was extracted using HP Total RNA Kit according to the manufacturers proto col.

Two micrograms of RNA were reverse transcribed using Superscript II RT according to the manufacturers protocol. Fast Start Universal SYBR Green Master Mix was used to perform qPCR for CYP1A1 on a BioRad CFX 96 instrument, using RPL13A as a housekeeping Inhibitors,Modulators,Libraries gene. The primer sequences Propidum iodide staining AFs ability to alter the cell cycle in MDA MB 468shAhR and Cal51shAhR cells was analyzed using a propidium iodide staining assay according to manufacturers pro tocols. Briefly, MDA MB 468shAhR cells were seeded into six well tissue culture plates and treated with 0. 1% DMSO or Inhibitors,Modulators,Libraries 25nM AF for 4, 24, 48, 72, or 120 hours. Cal51shAhR cells were seeded into six well tissue culture plates and treated with 0. 1% DMSO or 250nM AF for 24, 48, 72, 120, or 168 hours. Triplicate samples were col lected for all controls, and duplicate samples were col lected for all treatment groups.

Cells were harvested and fixed with selleck inhibitor EtOH up to a concentration of 70%, and kept at 4 C until PI staining. Samples were then analyzed by a FACScalibur instrument for cell cycle alterations. Data was analyzed using ModFitLT 3. 2. 1. Analysis of apoptosis and DNA damage AFs ability to induce apoptosis and DNA damage in MDA MB 468 and Cal51 cells was analyzed using an Apoptosis, DNA Damage, and Cell Proliferation flow cytometry kit, according to the manufac turers protocol.