Cell lines and cell culture Maintenance of the human Compound C Inhibitors,Modulators,Libraries PDAC cell lines PANC 1 and COLO 357 was described earlier. PANC 1 cells stably transduced with dn Rac1 retroviral vectors were cultured in the presence of 2. 5 ug/ml puromycin. RNA isolation and RT PCR analysis Total RNA from PANC 1 cells was isolated with peq GOLD RNAPure and reverse transcribed using Superscript II Reverse Tran scriptase. The primer sequences for BGN, b actin, MMP 2, and TATA box binding protein were given earlier. The mRNA expression was quantified by quantitative real time RT PCR on an I Cycler with I Cycler software. SYBR green was used for detection of amplification products. All values for BGN and MMP 2 mRNA concentrations were normalized to those for b actin and TBP specific transcripts in the same sample to account for small differences in cDNA input.

Construction of vectors and retroviral infection The construction of a retroviral vector for human dn Rac1 and of pcDNA3 based expression Inhibitors,Modulators,Libraries vectors for FLAG tagged Inhibitors,Modulators,Libraries Smad2 and GADD45b was described Inhibitors,Modulators,Libraries previously. A cDNA insert of a MYC tagged version of dn Rac1 was released from the pRK5 MYC vector and subcloned in pcDNA3. Transient transfections of expression vectors and siRNAs and reporter gene assays For transient transfections followed by immunoprecipi tation, PANC 1 cells were seeded at a density of 2 104 cells/cm2 in 6 cm plates on day 1, and on day 2 were co transfected serum free with Lipofectamine Plus according to the manufacturers instructions with FLAG tagged Smad2 in combination with either empty pcDNA3 vector, HA tagged FRNK, MYC tagged dn Rac1, or MYC tagged ca Rac1 as indicated in the legend to Figure 7.

Following removal of the transfection solution and a recovery period Inhibitors,Modulators,Libraries of 24 h in normal growth medium, cells were stimulated with TGF b1 for 1 h. The transfected cells were then lysed in IP buffer and pro cessed for anti FLAG, anti HA, and anti MYC immuno precipitation and immunoblotting. SiRNAs specific for Rac1 and matched negative control were purchased from Thermo Scientific Dharmacon, while prevalidated siRNAs to Smad2 and Smad3 as well as matched control were from Qiagen. Rac1, Smad2/3, and negative control siRNAs were transfected twice on two consecutive days with either Lipofectamine 2000 or Lipofectamine RNAi Max and HiperFect according to the suppliers recommenda tions.

For reporter gene assays, cells were seeded in 96 well plates and were co transfected on the next day serum free etc with either Lipofectamine Plus or Lipofecta mine 2000 with various cDNAs at an equal molar ratio together with dn Rac1 and either pAR3 luc FAST 1, or pCAGA luc, along with the Renilla luciferase encoding vector pRL TK. Each well received the same total amount of DNA and empty vector was added as needed. Following transfection and TGF b1 stimulation, luciferase activities were determined with the Dual Luciferase Assay System.