Figure 3
Antiapoptotic effects of CXCL12 treatment on gemcitabine-induced cell death. (A) DNA fragmentation assay. Cells were seeded in 6-cm Petri dishes and treated with 5 and 10 μ M gemcitabine in the absence or presence of CXCL12 (100 ng ml−1) for 48 h. Subsequently, genomic DNA was isolated and resolved (2 μg per lane) on 1% agarose gel. Lane 1: untreated, lanes 2 and 3: gemcitabine-treated (5 and 10 μ M), respectively, and lanes 4 and 5: gemcitabine-treated (5 and 10 μ M, respectively) in the presence of CXCL12. CXCL12-treated pancreatic cancer cells exhibit reduced DNA laddering compared with cells treated with gemcitabine only. (B) In situ determination of apoptosis. Panc1 and MiaPaCa cells were cultured on chamber slides and treated with gemcitabine (5 μ M) in the absence and presence of CXCL12 (100 ng ml-1). Apoptosis was detected by staining the cells with CaspACE FITC-VAD-FMK solution in PBS for 2 h at 37°C. Following fixation, bound marker was visualised by fluorescent detection under a confocal microscope. Representative pictures (overlay of FITC and DAPI) are from one of the random fields of untreated, gemcitabine only, and gemcitabine+CXCL12-treated Panc1 and MiaPaCa cells. Apoptotic cells that stained positively with FITC-labelled marker were counted in 10 random fields and presented in a bar diagram (mean±s.d.). *Significant difference as compared with gemcitabine only-treated cells. CXCL12 co-treated cells exhibited 53 and 55% reduced apoptosis by gemcitabine in Panc1 and MiaPaCa cells, respectively.
