Figure 4
Identification of modulated gene expression caused by Aurora-A expression during G0/G1 progression. 24 hours after serum removal, arrested Wi-38 cells were infected with adenoviruses expressing either Aurora-A or lacZ (control). Serum was added 48 hours after infection and cells were harvested at the times indicated. Total RNA was extracted and Clonetech human Atlas 1.2 cDNA expression array analyses were performed. (A) A list of the genes, which fail to respond to serum addition in Aurora-A overexpressing cells is presented in the upper panel. Ratio given indicates the difference in mRNA expression compared to controls. The lower panel highlights the region of the array containing the probes for Cyclin D1 and p19INK4d. (B) Verification of the array data was done using Northern blot analysis with a Cyclin D1 probe spanning the first 350 nucleotides of the Cyclin D1 coding sequence and a probe for GAPDH from nucleotides 192 to 549 for normalization. One of 3 Northern blots from independent experiments is shown. The expression level ratios of Cyclin D1 were calculated after densitometric analysis using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and normalized to GAPDH. Serum-deprived control cells were set to 1. (C) Immunoblot of equally treated cells was performed and assayed for Cyclin D1 protein expression. (D) At the indicated times, immunoblotting of serum released cells expressing either lacZ (control) or Aurora-A was performed with the antibodies indicated.