Figure 1. Cyclin D1 controls cell adhesion, cell migration, and cytokine production.

(A) 96-well plates were coated with fibronectin or HS-5 stromal cells. GFP- and D1-GFP-expressing clones were stained with calcein-AM and seeded. After incubation for 3 or 24 h at 37°C, non-adherent cells were removed by extensive washing. The plates were read with the Victor ×4 plate-reader. The percentage of cell adhesion was calculated by the ratio fluorescence of adherent cells/fluorescence of total cells x 100. Presented results corresponded to the mean of four independent experiments with five replicates. (B) GFP- and D1-GFP expressing clones were seeded in the top chamber of transwell inserts. The inserts were then placed in culture medium with FCS (+) or without FCS (−) as a control for specificity. The cells were incubated for 4 h at 37°C, and the number of migrating cells within the bottom of the insert was counted by flow cytometry. The results presented correspond to the mean of three independent experiments performed in triplicate. (C) GFP- and D1-GFP-expressing clones were cytospun on glass slides, stained with rhodamine-stained phalloidin for visualizing F-actin and counterstained with DAPI. The slides were analyzed with a confocal microscope (×180, magnification). (D) The Cytokine Array kit (panel A) was used for the detection of cytokines secreted in the culture medium by GFP- and D1-GFP clones. The assay procedure was performed according to the manufacturer's instructions. Spots corresponding to positive controls (C+), negative controls (C−), and produced cytokines are circled. *p < 0.05 with the t-test.