FIGURE 6.

CB₂ cannabinoid receptor-induced HiB5 cell proliferation is mediated by p27Kip1 inhibition. A and B, wild-type embryonic E14.5 cortical slices were treated for 1 h with vehicle (V) or HU-308 alone (HU; 5 μm) or in the presence of SR144528 (SR; 25 μm) or rapamycin (Rapa; 250 nm). Phospho-p27Kip1⁺ cells were quantified in the ventricular and subventricular zone (VZ/SVZ) after immunofluorescence and referred to the analyzed surface. Representative images are shown. C, HiB5 cells were treated with vehicle (Veh) or HU-308 (50 nm) for the indicated times, and p27Kip1 phosphorylation was determined by Western blot analysis. Loading control was performed with anti-α-tubulin antibody. D and E, HiB5 cells were treated for 30 min with vehicle or HU-308 (HU) alone or in the presence of LY294,002 (LY; 5 μm), Akt inhibitor 1 (Inh1; 5 μm), and rapamycin (Rapa; 50 nm). p27Kip1⁺ cells were quantified by immunofluorescence in each condition. Representative images are shown. Results correspond to three independent experiments. Scale bars, B and E, 100 and 10 μm, respectively. **, p < 0.01 versus vehicle-treated cells or slices; #, p < 0.05; ##, p < 0.01 versus HU-308-treated cells or slices.