Fig. 2

PMA significantly induces overexpression of PKCα, p-PKCα and NF-κB p65 nuclear translocation in bladder cancer cell lines. a 5637 and T24 cells were treated with PMA (10 ng/ml) for 0, 15, 30, 60, and 240 min, and the total, nuclear and cytoplasmic proteins were extracted at the indicated time point; p-PKCα and nuclear/cytoplasmic p65 were measured by western blot. b Normalized protein expression levels were calculated and analyzed. The gels were run under the same experimental conditions. The band intensities were calculated using the ImageJ 1.46r software. β-Tubulin was used as an internal control for the total protein measurement, and Histone was used as a nucleoprotein reference. The ratio of the target gene to β-Tubulin/Histone was used to conduct the statistical analysis. *P < 0.05 and **P < 0.01, as determined by Student’s T-test. c Cells were treated with DMSO or PMA (10 ng/ml) for 1 h, and p65 localization was detected by immunofluorescence. The cells with nuclear translocation of p65 are indicated with red arrows for the 5637 and T24 cell lines. For the BIU-87 cell line, nuclear translocation of p65 is evident in almost all cells within the visual field after the PMA treatment, and p65 expression can be observed in both the cytosol and nucleus. Original magnification: 400×. Comparisons between the control and PMA groups were made based on the statistical analysis of the cells with nuclear localization of p65 counted in three random fields