Figure 6

Cannabinoid inhibits proliferation of U87MG cells. (A) Serum-starved U87MG cells were treated with 3 μM Hu210 with or without 30 ng/ml of PTX. After labeling with 10 μM BrdU for 12 h, cells were subjected to the BrdU incorporation assay. Data are presented as the average ± SE of at least three independent experiments. *, p < 0.001, compared to the control level; **, p < 0.001, compared to the level of Hu210 alone. (B) Serum-starved cells were treated with 20 μM Win 55,212-2 for 1 h with or without 30 ng/ml PTX, and cell lysates immunoprecipitated with anti-Necdin antibodies. The precipitates were analyzed with antibodies against E2F1. Input was loaded with 5% of U87MG cell extracts used for the immunoprecipitation assay. (C) U87MG cells transfected with pGL3-E2F4B-Luc (0.3 μg) and pCMV-β-gal (0.3 μg) were treated with the indicated concentrations (μM) of Hu210 for 6 h with or without 30 ng/ml of PTX. Luciferase activity was normalized to that of β-galactosidase. Data are shown as the average ± SE of at least three independent experiments. *, p < 0.05; **, p < 0.001. (D) U87MG cells transfected with pcDNA3.1 or Necdin-shRNA were subjected to real-time PCR analysis. Data are shown as the average ± SE of at least three independent experiments. *, p < 0.05 compare to pcDNA3.1 level. (E) The cells transfected with pGL3-E2F4B-Luc (0.3 μg) and pCMV-β-gal (0.3 μg) or Necdin-shRNA (0.2 μg) were treated with indicated concentrations (μM) of Win 55,212-2 for 6 h. luciferase activity was normalized to that of β-galactosidase. Data are shown as the average ± SE of at least three independent experiments. *, p < 0.001.