We determined the induction of apoptosis by Poly polymerase cleavage and terminal deo ynucleotidyl transferase mediated dUTP nick end Inhibitors,Modulators,Libraries labeling assay. Autophagy was detected by monitoring the formation of microtubule associated protein 1A 1B light chain 3. LC3 consists 2 forms the cytosolic form LC3 I and the membrane bound form LC3 II. When autoph agy is induced, an increase of migrating band LC3 II can be seen by Western blotting. LC3 can also be detected by immunofluoresence. LC3 II stains with a punctate pattern whereas LC3 I has a diffused staining pattern. Forty eight hours after siRNA mediated Mcl 1 knock down, PARP cleavage was observed in MIA PaCa 2 cells, but not in S2 VP10 cells indicating that apoptosis occurs in MIA PaCa 2 cells.

however, LC3 II was present in S2 VP10 cells, but not in MIA PaCa Inhibitors,Modulators,Libraries 2 cells, indicating an onset of Carfilzomib autophagy in these cells. We used TUNEL to further confirm apoptotic cell death after Mcl 1 siRNA transfection. TUNEL positive cells were quantitated. Mcl 1 siRNA transfection significantly Inhibitors,Modulators,Libraries pro moted MIA PaCa 2 pancreatic cancer cells apoptosis. We also use LC3 immunofluorescence assay to detect autophagy in S2 VP10 pancreatic cancer cells after Mcl 1 siRNA transfection. A homogenous cytosolic distribution of LC3 can be detected in untreated S2 VP10 cells, which shifted to a punctate pattern after Mcl 1 siRNA transfection. We therefore conclude that siRNA mediated Mcl 1 knockdown induces pancreatic cancer death through apoptosis in MIA PaCa 2 cells and autophagy in S2 VP10 cells.

Mcl 1 is a target of miR 204 in pancreatic cancer cells Once we had established that Mcl 1 is required for pan creatic cancer cell survival, we investigated the mechanism of regulation of Mcl 1. Using TargetScan 6. 2, a database identifying putative miRNAs associated with mRNA, we identified Mcl 1 as a hypothetical target gene of miR 204. A previous study Inhibitors,Modulators,Libraries has shown that miR 204 is down regulated in head and neck cancer, but there is no information available on the e pression of miR 204 in pancreatic cancer cells. We therefore evaluated miR 204 e pression using real time PCR in different pancreatic cancer cell lines and compared it to a normal pancreatic ductal cell line. E pression of miR 204 was lower in all cancer cell lines evaluated, compared to HPDEC. Since miR 204 was inhibited in pancreatic cancer cells, we assessed the effect of its up regulation on cell sur vival.

For this, we first over e pressed the miR 204 mimic in MIA PaCa 2 and S2 VP10 cells. Compared to control miRNA, miR 204 levels increased by 33493 6754 and 27353 2520 fold 48 h post transfection in MIA PaCa 2 and S2 VP10 cells, respectively. Once we had established that miR 204 levels were increased in the presence of mimic, we assessed cell viability in the presence of the mimic.