05 gl Hoechst 33258 dye in PBS for 30 minutes at room temperature, washed three times with PBS and, finally, mounted in 50% glycerol in PBS, as previously described. Fluorescent nuclei with apop totic characteristics were detected by microscopy under ultra violet illumination at 365 nm. The images were photographed with a Nikon Coolpix 5000 and digitalised. For differential cell counting at least 500 cells were analysed. Immunoblotting Acinar cells were homogenised at 4 C in 50 mM Tris HCl buffer at pH 7. 5 with 0. 15% Triton X 100 and protease inhibitors as previously reported. Once centrifuged at 5000 g for 10 minutes at 4 C, superna tants were frozen at 80 C until used and an aliquot of each sample was separated for protein determination.
Extracts were subjected to 10% SDS PAGE, trans ferred onto nitrocellulose membranes and immunoblotted with rabbit polyclonal anti Bax, rat monoclonal anti TP53INP1 or goat polyclonal anti actin used as primary antibodies. Mem branes were incubated overnight and revealed selleckchem with peroxi dase conjugated secondary antibodies followed by enhanced chemiluminescence detection system. Densitometric analysis of protein levels was performed with ImageQuant software. RNA extraction and PCR amplification of cDNA Total RNA was extracted from acinar cells with Trizol as described. Reverse transcribed cDNAs were amplified using specific primers for Bax, TP53INP1s, TNFR1 and BclxL with glyceraldehyde 3 phos phate dehydrogenase primers serving as an internal control. GAPDH is considered an appropriate housekeeping gene in this model.
were used as forward and reverse primers, respectively. The reaction yielded a cDNA fragment 394 base pairs in length. For TP53INP1 forward 5 and TP53INP1 reverse TATC 3. After denaturing for three min utes at 96 C, 30 cycles of amplification using a step program, buy inhibitor 55 C, 30 seconds and 72 C, 1 minute and a final extension at 72 C 10 minutes was performed. PCR conditions for VPAC1 VPAC2 were 94 C for 10 minutes, 35 cycles of 94 C for 45 seconds, 55 C for 45 seconds, 72 C for 90 seconds and 72 C for 10 minutes while for Bax were 96 C for 3 minutes, 30 cycles of 58 C for 30 seconds, 72 C for 1 minute and 72 C for 10 minutes. PCR, products were size fractionated on 2% agarose gels and visualised by staining with ethidium bro mide using a size molecular marker. Scion Image for Windows program for processing.
The analysis of images was used to measure areas and the ratio of interest gene vs. housekeeping gene is depicted in each graph. Caspase 3 activity After appropriate treatments, cells were harvested by centrifu gation at 1000 g for five minute at 4 C. Cell pellets were washed with 1 ml of PBS, then suspended in 150l of lysis buffer containing 1 mM phenylmethylsulphonyl flu oride protease inhibitor and maintained for 30 minutes on ice.