Each PCR reaction was performed in triplicate in 50 µl with 600 µM (final concentration) each of the forward and reverse primers, and 25 µl of 2× SYBR green PCR Master Mix ABII) with the following profile: one cycle at 50°C for 2 min, one cycle at 95°C for 10 min, and then 40 cycles of 95°C for 15 s, 60°C for 1 min (two-step protocol).
Cells were preserved in a 1% final concentration each of formaldehyde and glutaraldehyde buffered with sterile f/2 medium.
The IC50 value for curcumin was within the 8-10 micrangeange for inhibiting the growth of T47D cells induced by a 10- microM concentration each of 4-nonylphenol, 4-octylphenol, and o, p'-DDT.
Joint tissues were homogenized on ice in 3-ml lysis buffer (PBS containing 2 m M PMSF, and 0.1 mg/ml (final concentration), each of aprotinin, antipain, leupeptin, and pepstatin A) by using Polytron (Brinkinarm Instruments, Westbury, NY, USA).
Each reaction (20 μl) contained 5 μl of the diluted cDNA (equivalent to 2.5 ng of total RNA) and 300 nM final concentration each of forward and reverse primer.
The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μmol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St . Louis MO, USA).
