Figure 2.
Inhibitory effects of sCD95Fc on the gemcitabine-induced expression of PICs (IL8 and IL6), and PCSC markers (CD44 and CD133) in pancreatic tumor cells. Human PDAC cell line PancTuI-luc was seeded with 2.8 × 105 cells/well in a 6-well plate. Cells were either treated with 0.9 % saline (A–C) or untreated (D) as controls or treated with gemcitabine (20 μM) alone (A,C) or in combination with three different concentrations of sCD95Fc (20, 60, 120 μg/mL) (D). Cells were lysed in RNA Lysis buffer T, total RNA was isolated, and assayed for CD95 and CD95L (A, upper row) or IL8, IL6, CD44 and CD133 (C,D) via PerfectProbe real-time RT-PCR assays. Relative expression was normalized to the control and reference gene UBC and expressed as arbitrary unit (AU). Controls were set as 1 in each experiment. Data represent means ± SD of biological replicates (n = 2) with three technical replicates each. In case of statistical significance, asterisks indicate the corresponding significance levels (Student’s t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant). (A,C,D). B: mCD95 and mCD95L cell surface expression was assayed in non-permeabilized PancTuI-luc using phycoerythrin-conjugated mouse anti-human-CD95 antibody DX-2 (filled yellow peak-untreated and Red line peaks-treated with Gemcitabine (20 μM) and mouse anti-human-CD95L antibody NOK-1 (black line peaks-untreated and red line peaks -treated with gemcitabine (20 μM) or an irrelevant antibody (IgGl isotype control) used for the determination of background staining (grey filled peaks-untreated and green line peaks-treated with gemcitabine (20 μM). Raw data were evaluated as histogram plots as described in Section 2.