Fig. 1.

a Proliferation of MIAPaCa2 cells in medium containing various concentrations of GEM. MIAPaCa2 cells (3 × 10⁵/well) were seeded in 6-well culture plates in regular culture medium, which was then exchanged for GEM-containing medium after 24 h. At 24-h intervals, cells were detached using trypsin, and cell numbers were counted using a hemocytometer (n = 3). b Up-regulation of WT1 mRNA in MIAPaCa2 cells by GEM treatment. Twenty-four hours after plating, culture medium was exchanged to media containing GEM at indicated concentrations (0, 10, 30 and 100 ng/ml). MIAPaCa2 cells were harvested at 24-h intervals, and WT1 mRNA in cell homogenates was analyzed using qRT-PCR. WT1mRNA levels were normalized relative to those of 18S ribosomal RNA (18S). c Up-regulation of WT1 mRNA in MIAPaCa2 cells after short treatment with GEM. Twenty-four hours after plating, MIAPaCa2 cells were untreated or treated with 100 ng/ml of GEM for 2 h. MIAPaCa2 cells did not proliferate but kept alive for following 72 h by this treatment with GEM. After GEM treatment, cells were washed well, cultured in regular culture medium, and harvested at 24-h intervals. WT1 mRNA in cell homogenates was analyzed using qRT-PCR, and WT1mRNA levels were normalized relative to those of 18S ribosomal RNA (18S). d NF-kB suppresses GEM-induced up-regulation of WT1 mRNA. MIAPaCa2 cells (6 × 10⁴/well) were seeded in 24-well culture plates. After 24 h, medium was exchanged for media containing GEM (0 or 30 ng/ml) and/or NF-kB blocking peptide (50 μM) or control peptide (50 μM). WT1 mRNA levels were quantified after 24-h incubation using qRT-PCR. *P < 0.01