FIG. 5.

The cdk6 cytoplasmic-to-nuclear trafficking with live cell imaging. A: Schematic representation of the Ad.cdk6-GFP construct used in the live cell imaging experiments, with a GFP tag at the C-terminus. Numbers above indicate amino acid residues. The blue box designates the ATP-binding domain, the black box designates the INK4-binding domain, the red box designates the catalytic loop, and the two green boxes designate the activation loops. The cdk6 can be phosphorylated on Thr¹⁷⁷ by cdk-activating kinase (CAK). Key amino acids in the substrate-binding pocket are indicated. B: Representative immunoblots of human islets transduced with Ad.lacZ (control [CTL]), Ad.cdk-6-GFP, nontagged Ad.cdk6, with or without Ad.cyclin D3, showing phosphorylated Rb (phos-Rb), total Rb (Rb), GFP, cdk6, and cyclin D3, and tubulin as loading control. Small numbers on the left indicate molecular weight markers. These studies indicate that the Ad.cdk6-GFP is biologically active because it can phosphorylate pRb. C: BrdU-positive and insulin-positive cells as a percentage of total insulin-positive cells. Representative pictures of BrdU (red) and insulin (green) in the conditions are shown at the bottom of the panel. Dispersed human islets were transduced with control Ad.lacZ (CTL) or with adenoviruses encoding cdk6 (C6), cdk6-GFP (C6-GFP), cdk6 and cyclin D3 (C6+D3), or cdk6-GFP and cyclin D3 (C6-GFP+D3). The numbers below each bar indicate the numbers of different human islet preparations studied. Bars indicate mean ± SEM. *P < 0.05; ns, nonsignificant. These studies confirm that the Ad.cdk6-GFP is biologically active. D: Photomicrographs derived from live cell imaging of Ins1 cells transduced with cdk6-GFP (the full movie available as Supplementary Movie 1). E: Photomicrographs derived from live cell imaging of dispersed human islet cells transduced with cdk6-GFP. The numbers above each picture in D and E indicate the time from the beginning of the movie (the full movie available asSupplementary Movie 2). Ins, insulin.