Fig 7
The expression of both NF-κB and HIF-1α in GEM-R and GEM-S PaCa cells. (a) The expression of NF-κB in GEM-R/S PaCa cells. The activity of NF-κB in GEM-R and GEM-S MIA PaCa-2 cells treated without GEM was measured by NF-κB (p65) transcription factor assay. Values are expressed as means ± SD. Between-group statistical significance was determined using the Student’s t test. **, P < 0.01. (b) The effect of GEM on NF-κB activity in GEM-R PaCa cells. GEM-R MIA PaCa-2 cells were treated with different concentrations of GEM (0–20 μM) for 72 h. The NF-κB p65 protein levels in GEM-R were measured. Values are expressed as means ± SD. Multiple comparisons were performed by using one-way ANOVA followed by Dunnett’s test. **, P < 0.01; *, P < 0.05 versus control (0 μM). (c) The expression of HIF-1α in implanted PaCa tumor. The expression of HIF-1α protein determined by immunohistochemical staining (brown, HIF-1α protein, blue, nucleus) in implanted PaCa tumor: (c-1) GEM-S, (c-2) GEM-S treated with GEM, (c-3) GEM-R, (c-4) GEM-R treated with GEM. Original magnification, ×1000, scale bars, 20 μm. (d) Quantification of immunostaining of HIF-1α protein by digital image analysis. For each image, the color deconvolution method was used to isolate HIF-1α-positive DAB-stained cells from HIF-1α-negative hematoxylin-stained cells. The measurement parameter was IOD. Optical density was calibrated and the area of interest was set as follows: hue, 0–30; saturation, 0–255; intensity, 0–255. The values were determined, and the IOD was log10 transformed. Values are expressed as means ± SD. Multiple comparisons were performed using one-way ANOVA followed by Bonferroni test, **, P < 0.01; *, P < 0.05