H520 cells have increased levels of the Cdk5 activator p39.

(A) Immunoblots showing comparable levels of Cdk5 and p35 in H1666 and H520 cells. However, H520 cells have increased expression of the second major Cdk5 activator p39, relative to H1666 cells. Alpha-beta tubulin was blotted as a loading control. (B) After quantification of band intensity in triplicate immunoblots only p39 expression resulted to be increased in a statistically significant manner in H520 cells relative to H1666 cells (p-value of 0.007) based on an unpaired t-test statistical analysis. (C) Immunoblots showing that phosphorylation levels of Rb S249 and T821 were not significantly affected upon reintroduction of p39 into H1666 cells, in spite of a robust p39 expression in H1666 cells 24 hours after adeno-p39 infection relative to cells transfected with an adenovirus bearing a scrambled p39 sequence. (D) Band intensities in the triplicate immunoblots shown in C. were quantified using the ImageJ Plug-in and phosphorylation in Rb S249 and T821 was graphed after normalization against total Rb. No significant effect on Rb phosphorylation on S249 or T821 upon p39 forced expression in H1666 cells can be detected, with p-values shown at the top of each graph. (E) NSCLC microarray expression data from the Oncomine database (http://www.oncomine.org, Compendia Bioscience, Ann Arbor, MI, USA), specifically from the study conducted by Bhatacharjee et al. This study contained 139 lung adenocarcinomas, 21 squamous cell carcinomas, 6 small cell lung carcinomas, and 17 normal lung tissues. Datasets were ordered by overexpression of CDK5R2 (p39), which was analyzed by Affymetrix U95A microarrays (Affymetrix ID 36361_at). Box plots show that p39 is over-expressed 1.76-fold in AC relative to normal lung (p-value of 0.060) and that the p39 overexpression is more pronounced in SCC by 2.71-fold relative to normal lung (p-value of 0.012). (F) Kaplan Meier plots showing reduced overall survival of AC patients with higher expression of p39 (p-value of 0.0013), the effect being less pronounced and non-statistically significant in SCC patients. The plots were generated using the KM Plotter Online Tool (http://www.kmplot.com), which relates microarray expression data (Affymetrix ID: 20582_at, in this case for p39 expression) with clinical outcomes. Statistical tests were assessed by Mann-Whitney tests when comparing two groups and Kruskal Wallis when comparing three groups or more. Statistical significance was set as: *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001.