Cell lines were obtained from the Cell Resource Centre at Peking Union Medical College (PUMC), China. Panc-1 cells were cultured in Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% foetal bovine serum (FBS), and SW1990 cells were cultured in RPMI-1640 (HyClone) with 10% FBS in 5% CO₂ at 37°C. Adherent cells were passaged every 2–3 days with 0.5 mg/ml trypsin (1:250) and 0.53 mM ethylenediaminetetraacetic acid. c-Jun expression plasmids were cloned into pIRES-puro2 with a C-terminal Myc tag. siRNA oligonucleotides were designed against c-Jun as follows: GAU GGA AAC GAC CUU CUAU. The plasmids were constructed according to standard cloning techniques. Cells were transfected using Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA).

The Bcl-3 and Bim antibodies were purchased from Santa Cruz Biotechnology. The antibody against c-Jun was purchased from BD Pharmingen (San Diego, CA, USA). Antibodies against phospho-c-Jun (Ser73), Bax, PARP, caspase-7 and GAPDH were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, MA, USA).

Proteins were extracted with sodium dodecyl sulphate (SDS) lysis buffer [50 mM Tris-HCl (pH 6.8), 10% glycerol and 2% SDS] and quantified using the bicinchoninic acid protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA). Extracts were separated on a 12% SDS-polyacrylamide gel and electrophoretically transferred to polyvinylidene fluoride membrane (GE Healthcare Life Sciences). The membrane was blocked in 5% skimmed milk for 1 h at room temperature and then incubated overnight with the indicated antibodies at 4°C. The membrane was incubated with an anti-rabbit or an anti-mouse IgG-HRP (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Chemiluminescence was detected using an enhanced chemiluminescence blot detection system (Santa Cruz Biotechnology, Inc.).

Total RNA from cells was extracted with TRIzol reagent (Invitrogen Life Technologies) and 1 µg isolated total RNA was converted to cDNA using a First-Strand cDNA synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China). Power SYBR-Green master mix (Applied Biosystems, Foster City, CA, USA) was added to cDNA samples that were then subjected to RT-qPCR using the StepOne Real-Time PCR system (Applied Biosystems). Relative mRNA levels were normalised against the housekeeping gene GAPDH. The primers for RT-qPCR were as follows: c-Jun sense, 5′-TCCAAGTGCCGAAAAAGGAAG-3′ and antisense, 5′-CGAGTTCTGAGCTTTCAAGGT-3′; c-Fos sense, 5′-GGGGCAAGGTGGAACAGTTAT-3′ and antisense, 5′-CCGCTTGGAGTGTATCAGTCA-3′; GAPDH sense, 5′-TGAGTACGTCGTGGAGTCCA-3′, and antisense, 5′-TAGACTCCACGACATACTCA-3′.

Cell proliferation was assessed by the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Shanghai, China). Cells were seeded at a density of 2,000 cells/well in 96-well plates. A total of 10 µl CCK-8 solution was added to each well containing 100 µl culture medium and incubated for 2 h at 37°C. Absorbance was measured at 450 nm using a multiwell spectrophotometer (BioTek, Winooski, VT, USA).

Cells were harvested by trypsinisation and collected by centrifugation, and then a fluorescein isothiocyanate (FITC)-Annexin V kit (NeoBioscience, Shenzhen, China) was used to stain cells according to the manufacturer's instructions. Apoptotic cells were analysed with a BD Accuri^® C6 flow cytometer and corresponding CellFIT software (both from BD Biosciences, San Diego, CA, USA).

Cells were fixed with 4% paraformaldehyde solution for 30 min at room temperature. After rinsing with phosphate-buffered saline (PBS), the samples were incubated with a TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and FITC-dUTP (Roche Applied Science, Indianapolis, IN, USA) for 1 h at 37 °C using an apoptosis detection kit (Roche Applied Science). These cells were then stained with 4,6-diamidino-2-phenylindole (DAPI) to detect the cell nucleus.

Statistical analyses were performed using Student's t-test in Microsoft Excel software (Redmont, WA, USA). The results were presented as the means ± standard deviation of triplicates of each experiment. All experiments were performed three times, unless stated otherwise.

To determine the physiologically relevant dose of gemcitabine that induces apoptosis in Panc-1 and SW1990 cells, we first determined gemcitabine's cytotoxic effects and used 293A cells as a control. We treated cells with increasing doses of gemcitabine (from 0.1 to 1 mM) or PBS for control cells. The results identified the 50% inhibitory concentration (IC₅₀) value in Panc-1 and SW1990 cells (Fig. 1A and B) while 293A cells did not respond to gemcitabine (Fig. 1C). Based on the data and previous studies reporting that micromolar gemcitabine concentrations may be clinically achieved (20), we used 10 µM gemcitabine for all subsequent experiments. Flow cytometric analysis revealed that 10 µM gemcitabine significantly increased apoptosis in Panc-1 and SW1990 cells (Fig. 1D and E), but not in 293A cells (Fig. 1F).

c-Jun and c-Fos are extensively studied components of the AP-1 complex, which is involved in numerous cell activities, including proliferation, apoptosis, survival, tumourigenesis and tissue morphogenesis (21). To further define the AP-1 mechanism in gemcitabine-induced pancreatic cancer cell apoptosis, we examined the expression of c-Jun and c-Fos following exposure to 10 µM gemcitabine at the indicated time points through western blot analysis and RT-qPCR. As shown in Fig. 2A and B, gemcitabine had little effect on c-Fos expression but significantly increased c-Jun expression. These results suggest that gemcitabine specifically activates the AP-1 pathway through c-Jun but not c-Fos. In addition, gemcitabine increased c-Jun expression in a concentration-dependent manner (Fig. 2C and D).

Based on the experimental data above, we hypothesised that c-Jun regulates gemcitabine-induced apoptosis. To confirm this hypothesis, we transfected c-Jun (c-Jun-Myc) or a control vector into Panc-1 and SW1990 cells for 24 h. We then exposed the cells to gemcitabine and performed fluorescence-activated cell sorting (FACS) analysis to measure apoptosis at the indicated time points. The results revealed that gemcitabine-induced apoptosis increased upon c-Jun overexpression (Fig. 3A and B). Western blot analysis also suggested that PARP and cleaved caspase 7 levels increased, indicative of the increased apoptosis. AP-1 likely affects apoptosis through the differential regulation of pro-apoptotic and anti-apoptotic downstream factors (22,23). Therefore, we analysed Bcl-3, Bax and Bim expression. As shown in Fig. 4A and B, Bcl-3 and Bax expression did not change upon c-Jun overexpression, whereas Bim expression increased. Therefore, c-Jun overexpression promotes gemcitabine-induced apoptosis though Bim upregulation.

To further determine the pro-apoptotic properties of c-Jun in gemcitabine resistance, we suppressed endogenous c-Jun expression in Panc-1 cells by transfecting cells with siRNA against c-Jun. FACS and western blot analysis indicated that c-Jun knockdown inhibited gemcitabine-induced apoptosis (Fig. 5A). We also observed that Bim expression decreased following gemcitabine treatment in c-Jun knockdown cells. However, c-Jun did not affect Bax and Bcl-3 expression under the same conditions (Fig. 5B). Taken together, our findings suggest that c-Jun exerts pro-apoptotic effects on gemcitabine-treated cells by regulating its downstream target Bim in pancreatic cancer cells.

In addition to the expression level, the activity of c-Jun is also critical for its function. Therefore, we treated cells with SP600125, which blocks c-Jun phosphorylation. We exposed Panc-1 cells to gemcitabine for 24 h in the presence or absence of 20 µM (24) SP600125 and examined apoptosis by FACS analysis. The results revealed that SP600125 alone had little effect on Panc-1 cells, but SP600125 pre-treatment partially decreased gemcitabine-mediated apoptosis (Fig. 6A). TUNEL staining was used to further confirm the apoptosis results and the data also indicated that SP600125 pre-treatment partially decreased gemcitabine-mediated apoptosis (Fig. 6B). SP600125 attenuated PARP and cleaved caspase 7 levels (Fig. 6C). These results suggest that c-Jun activation is required for gemcitabine-induced apoptosis. Moreover, compared with the uniform Bcl-3 and Bax expression, a gemcitabine-induced increase in Bim expression was attenuated upon SP600125 treatment.