EXPERIMENTAL PROCEDURES Cell Culture and Transient Transfection Human breast cancer cell lines MDA-MB-435 and MDA-MB-231 and pancreatic cancer cell lines IMIM-PC1, Suit-028, and PaTu8988t were maintained in Dulbecco’s modified minimal essential medium (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% FCS. Transient transfection was performed by using TransFast selleck chemical reagent (Promega, Madison WI). Small interfering RNAs to human NFATc2 (siRNA 1: 5��-gcugaugagcggauccuuatt-3��; siRNA 2: 5��-ccauuaaacaggagcagaatt-3��) obtained from Ambion Applied Biosystems (Austin, TX), HDM2 (siRNA 1: 5��-ccacaaaucugauaguauuu-3��; siRNA 2 5��-gaugagguauaucaaguuauu-3��), or HDM2 (SMARTpool siRNA) and GSK-3�� (SMARTpool siRNA) obtained from Dharmacon (Lafayette, CO) were transfected into the indicated cell lines by using siLentFectTM (Bio-Rad) or Effectene? transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

ZOL was obtained from Novartis Oncology (Basel, Switzerland). Generation of Viral Vectors and Stable Cell Lines LinxA cells were purchased from Open Biosystems (Huntsville, AL) and were maintained in Dulbecco’s modified minimal essential medium (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% FCS and 100 ��g/ml hygromycin (Carl Roth GmbH, Karlsruhe, Germany). For retroviral expression, HA-tagged wt-NFATc2, as well as the mutated HA-NFATc2 pSP2 construct, were cloned into a pQCXIP vector (BD Biosciences, Heidelberg, Germany).

For retroviral production, the packaging cell line LinxA was transfected with 5 ��g of the retroviral plasmids in a 10-cm dish using LipofectamineTM 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. 48 and 72 h after transfection, the supernatant containing retroviral particles was filtered through a 0.45-��m filter and supplemented with 2 ��g/ml Polybrene (Millipore, Billerica, MA) to infect the target cells PaTu8988t and Suit-028. Spin infection was carried out at 2500 �� g and at 37 ��C. Transduced cells were selected with 1 ��g/ml puromycin (Sigma-Aldrich) for at least 2 weeks. The successful overexpression of the NFATc2 constructs was tested by immunoblotting. Plasmid Constructs All murine NFATc2 constructs were generated in pcDNA3.1 (+) HA tag vector by subcloning of the wt-NFATc2 open reading frame into HindIII and XbaI. The Anacetrapib ��SP2 mutation was generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) by introducing single-point mutations at serines Ser-215, Ser-219, and Ser-223 (corresponding to human NFATc2 serines at Ser-213, Ser-215, and Ser-221), respectively.