Figure 7

Inhibition of Aurora A and effect on ciliary formation in cancer OSE cells. A) Results of three independent siRNA knock-down experiments of AURA in SK-OV3 cells. Cells were grown to 60% confluency and transfected with scrambled oligonucleotides (mock) or siRNA targeting AURA mRNA. The cells were allowed to grow to confluence and 24 hours after transfection, medium was changed to serum-depleted medium. After 72 hours of incubation in serum-depleted medium, AURA protein levels were examined by WB analysis. Anti-phosphorylated retinoblastoma protein (p-RB) was included to explore the cell cycle stage. EB1 was applied as loading control. Percentages of ciliated cells were determined by IFM analysis with primary antibodies against acetylated α-tubulin, detyrosinated α-tubulin, and DAPI nuclear staining as the number of ciliated cells over the total cell number. Error bars represent standard deviations. B) Bar graph representation of the level of ciliated SK-OV3 cells in AURA siRNA transfected cells relative to mock transfected cells. The percentage of ciliated cells in mock transfected cells was set to one. Data were tested for significance using Student’s t-test. The level of significance was set at P < 0.001 (***). C) Protein levels of the full length activator form of GLI2, GLI2 (FL), acetylated α-tubulin (Acet.tub), and α-tubulin (α-tub) in mock and AURA siRNA transfected SK-OV3 cells serum-depleted for 72 hours. D) Bar graph representation of the relative protein levels in AURA siRNA transfected SK-OV3 cells compared to mock transfected cells. The protein level in mock transfected cells was set to one. Data were tested for significance using Student’s t-test. The effect of AURA siRNA treatment on Acet.tub and α-tub protein levels was found to be not significant (n.s.), whereas the treatment had a significant effect on GLI2 (FL), P < 0.01 (**). DAPI, 4‘,6-diamidino-2-phenylindole; IFM, immunofluorescence microscopy; OSE, ovarian surface epithelium; WB, western blot.