Luciferase and renilla plasmids

In 12-well plates, HC-04
cells were plated in DMEM/F12 media with 10% FBS and 1% penicillin–streptomycin
and MCF-7 cells were plated in RPMI 1640 media without phenol red
and supplemented with 10% charcoal-stripped FBS, 1% glutaMAX, 1% AB/AM,
1% nonessential amino acids, and insulin (6 ng/mL) overnight. Cells
were transfected at 70% confluency with luciferase and renilla plasmids
(Promega, Madison, WI), XRE pGL4.43, and pRL-TK, respectively, using
Lipofectamine 2000 reagent for 6 h. Cells were treated with the RCE/isoflavones
for 24 h and lysed with buffer. The lysates were analyzed for luciferase
activity according to Promega’s dual-luciferase reporter assay
system protocol using BioTek (Winooski, VT) synergy H4 hybrid multi-mode
microplate reader.^{6 (link)} For experiments with
ICI 182,780 in MCF-7 cells, the cells were pretreated with ICI 182,780
for 2 h and incubated with compounds for an additional 24 h before
the cells were lysed and analyzed for luciferase activity.

HepG2
and MCF-7 cells
were plated in 12-well plates overnight, and cells were transfected
at 70% confluency with luciferase and renilla plasmids (Promega, Madison,
WI), XRE pGL4.43 luciferase plasmid (1 μg), and pRL-TK (500
ng), using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY)
for 6 h. After 6 h of transfection, cells were treated with hop extract/compounds
with and without the presence of TCDD (10 nM) for 24 h and lysed with
passive lysis buffer. Lysates were centrifuged and analyzed for luciferase
activity according to Promega’s Dual-luciferase Reporter Assay
System protocol using the FLUOstar OPTIMA luminometer (BMG Labtechnologies,
Germany). The results were plotted as fold induction of the control.
The % of TCDD was obtained by setting TCDD’s fold induction
in the XRE-luciferase assay as 100%. The fold induction of compounds
was divided by the TCDD response and multiplied by 100 to obtain the
% of TCDD response.