Fig. 4. The nuclear form of cyclin D1 binds HIF1α.

a U266 cells were cultured under normoxia and analyzed by IF and confocal microscopy after DAPI (in blue) or HIF1α (in yellow) staining (×180 magnification). b LP1 cells were cultured under normoxia or treated with 300 μM CoCl₂ overnight and analyzed after DAPI (in blue) or HIF1α (in red) staining. Merged images of representative cells were processed with ImageJ software, and the curves of FI as a function of distance were exported. c D1b–GFP Cl2 cells were cultured under normoxia and analyzed by IF, as previously described. d Duolink PLA technology was used on D1b–GFP Cl2 cells treated with CoCl₂ (to enhance the signal) and U266 cells (as a control), to investigate cyclin D1/HIF1α and cyclin D1/CDK4 interactions, respectively. Slides were incubated with the primary Abs, except for the negative control (Ctrl−), and then with the secondary Abs conjugated to the MINUS and PLUS probes. The slides were counterstained with DAPI before confocal microscopy examination (×180 magnification). Representative fields (white squares) were enlarged (×3).