The human pancreatic adenocarcinoma cell lines PaTu 8988t were obtained from H. P. Elsässer (Philipps University of Marburg, Germany) and maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich) and 1% Normocyn (Amaxa biosystems). Cells were cultured at 37 °C in humidified CO2 atmosphere (5%) and maintained in monolayer culture. Experiments were done with cells at ~70–80% confluence.
Antibodies and reagents
Ionomycin was purchased from Sigma-Aldrich. For immunoblotting, membranes were probed with antibodies against NFATc2 (Santa cruz), Sp1 (Santa cruz), MEF 2A (Upstate cell signaling solutions), Lamin B (Santa cruz), and ß-actin (Sigma-Aldrich).
PaTu 8988t cells were grown on chambered coverslips, either left untreated or treated with 10% FCS, and subjected to transfection with the indicated plasmids (NFATc2-GFP). Cells were washed, fixed with 4% paraformaldehyde, blocked, and probed with NFATc2 (Santa cruz) and Sp1 (Santa cruz) antibodies. Proteins of interest were detected by means of fluorochrome-conjugated secondary antibodies (Invitrogene), and nuclei were counterstained with 4′6-diamino-2-phenylindole (DAPI). Cells were evaluated with a fluorescence microscope (Zeiss, Oberkochen, Germany).
Subcellular fractionation, co-immunoprecipitation, and immunoblotting
For subcellular fractionation, cells were washed twice with cold DPBS and re-suspended in lysis buffer (12.5 mL 1 M HEPES, ph 7.5, 7.5 mL 5 M NaCl, 1.25 mL 200 mM EGTA, 25 mL 100% Gycerin, 2.5 mL Triton X-100, 1.05 g NaF, 1.11 g Na4P2O7 × 10 H2O) containing the protease inhibitors Orthovandat (Sigma aldrich), Leupeptin (Sigma aldrich), Benzamidin (Sigma aldrich), PMSF (Sigma aldrich), Aprotinin (Sigma aldrich). After sonification, cells were centrifuged at 13.000 rpm for 5 min, and supernatants were transferred to new cups and incubated on ice.
For co-immunoprecipitation, 500 μg of the lysates was immunoprecipitated with 4 µL of the indicated antibodies and protein G or A agarose (Roche Diagnostics). The immunoprecipitates were subjected to immunoblotting.
For Western blotting, protein extracts were analyzed by SDS–PAGE and blotted onto nitrocellulose. Upon protein extraction and gel transfer, membranes were washed in TBS washing buffer and incubated with peroxidase-conjugated secondary antibodies. Immunoreactive proteins were visualized by means of an enhanced chemiluminescence detection system (Western blotting detection reagent, GE healthcare). Membranes were probed with anti-NFATc2 (Santa Cruz), anti-Sp1 (Santa cruz) and anti-MEF 2A (Upstate cell signaling solutions). Anti-Lamin B (Santa cruz) and anti-ß-actin (Sigma-Aldrich) antibodies were used as loading control.
DNA pull-down assay
For DNA pull-down assay, 200 μg proteins per sample was incubated for 3 h with 10 μL of biotinylated double-stranded oligonucleotides containing the GGAAA consensus NFAT binding sequence of the human interleukin-2 promoter (5′-tctaaggaggaaaaactgtttcatg-3′ and its complementary strand) (Biomers.net GmbH). DNA–protein complexes were further incubated with 60 µL streptavidin-agarose beads (Sigma-Aldrich) for 1 h, washed twice with lysis buffer, and subjected to immunoblotting.
Preparation of the recombinant GST-NFAT proteins and GST pull-down assay
Bacteriologically expressed GST fusion proteins were coded by pGEX plasmids. We used BL21 strains of Escherichia coli for amplification of the pGEX GST-NFAT plasmid and protein extraction. The transformed colonies were inoculated with 5 mL of LB medium (Roth) and 5 µL of ampicillin (Sigma-Aldrich), and the culture was incubated at 37 °C on an orbital shaker for 12–15 h (up to OD660 of 0.2–2.0). Expression of NFAT fusion proteins was induced by adding 0.75 mL of IPTG solution (AppliChem). Bacteria were lysed by sonification, and we identified the produced proteins by means of SDS–polyacrylamide gel electrophoresis. For the actual assay, we incubated 100 µL of purified glutathione agarose beads (GE Healthcare) with 3 µg of bacteriologically expressed GST or GST-NFAT and total protein at 4 °C for 15–18 h. After centrifugation and several wash cycles, samples were mixed with 30 µL of Laemmli puffer, heated up to 95 °C for 5 min, and analyzed by Western blotting.
Transient transfection, siRNA, and luciferase reporter assay
Cells were seeded in 12-well plates. For transient transfection of expression constructs, PaTu 8988t cells were transfected 24 h after seeding at 70% density using TransFast (Promega) as a transfection reagent according to the manufacturer’s instructions. The promoter constructs cisNFAT-Luc were kindly provided by Stratagene Garden Grove, USA.
Luciferase activity was measured using the Lumat LB 9501 (Berthold Technologies, Mannheim, Germany) luminometer and the dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Firefly luciferase values were normalized to Renilla luciferase activity and are shown as mean values ± SD.
For siRNA transfection, we obtained NFATc2 siRNA (5′-CCAUUAAACAGGAGCAGAAtt-3′), Sp1 siRNA (5′-GGUAGCUCUAAGUUUUGAUtt-3′), and the Silencer Negative Control from Ambion (applied biosystems). Cells were transfected for 24 h using the siLentFect lipid reagent (Biorad) according to the manufacturer’s protocol.