nevertheless Following washes in TBS T, blots were incu bated with the appropriate HRP labelled secondary anti body for 1 hr at room temperature. Visualization of protein bands was performed using the Supersignal West Pico Chemiluminescent Substrate exposed on Kodak film in a Konica Minolta SRX 101A tabletop processor. RT RNA isolation and RT PCR MCF 7 cells plated at 0. 8 106 cells per 10 cm dish were incubated at 37 C overnight. The next day cells were treated with either with M344, cisplatin or their combination for 24 hrs. Total RNA was extracted using the RNeasy1 kit. RNA con centrations were quantified using a NanoDrop ND 1000 spectrophotometer. One microgram of total RNA was reverse transcribed to complementary DNA for quantitative, real time, reverse transcriptase polymerase chain reaction as previously described.

The Applied Biosystems AB 7500 Real Time PCR system was used to detect amplification. Real time PCR reactions were carried out in a total volume of 25 ul that contained 2. 5 ul of synthesized cDNA, 1. 25 ul of TaqMan Gene Expression Assay Primer Probe, 12. 5 ul of TaqMan Universal PCR Master Mix and 8. 75 ul of RNase free water for ATF3 expression. The endogenous control for ATF3 was the housekeeping gene, human GAPDH. Amplification conditions were 95 C for 5 min, 40 PCR cycles at 95 C for 15 sec and 60 C for 1 min. Three independent experiments were performed to determine the average gene expression and standard deviation. Chromatin Immunoprecipitation Assay Cells treated for 24 hrs in 10 cm dishes were fixed with 1% formaldehyde for 20 min at room temperature in order to cross link the DNA and protein.

The cross linking was quenched by adding glycine to a final concentration of 200 mM and incubating at room temperature for 5 min. Cells were then washed twice with ice cold PBS and harvested in 1 mL cold PBS by centrifugation at 4 C for 5 min at 5,000 rpm. The pellet was resuspended in 90 uL lysis buffer supplemented with 1�� Protease Inhibitor Cocktail, 1 mM 1,4 dithio DL threitol, and 1 mM phenyl methylsulfonyl fluoride. The lysates were sonicated using a Sonicator 3000 at power setting 1 for a total of 3 min on ice with 10 sec on off pulses to shear the DNA to an average size of 300 to 1000 base pairs. Soni cated lysates were cleared of debris by centrifugation for 15 min at 14, 000rpm at 4 C. Input controls were removed from each sample and stored at 20 C.

Soni cated lysates were divided into negative controls and samples, then diluted 10 fold with dilution buffer supplemented with 1�� Protease Inhibitor Cocktail, 1 mM DTT, and 1 mM PMSF. Positive sample cell lysates were immunoprecipitated by overnight rotation at 4 C with rabbit anti acetyl H4 primary antibody. AV-951 Negative controls were incubated overnight with rotation at 4 C in the absence of primary antibody. Immune complexes were collected by 2 hr rotation at 4 C with the addition of 40 uL of protein A agarose sal mon sperm DNA 50% slurry to both samples and negative controls.