Figure 3.

HIF-1 was not involved in nickel-induced cyclin D1 or cyclin E expression. 8 × 10³ Beas-2B-Vc VEGF-luc mass1 and Beas-2B DN-HIF VEGF-luc mass1 cells (A) were seeded into each well of a 96-well plate. After being cultured at 37°C overnight, the cells were treated with NiCl₂ for various dosages as indicated. The cells were then extracted and luciferase activity was determined as described previously. (♣), a significant decrease as compared with nickel-treated Beas-2B VEGF-Luc mass1 cells (P < 0.05). Beas-2B Vector control cells, Beas-2B DN-HIF mass1 cells (B, C, D, and G) were seeded into 100-mm dishes. After being cultured at 37°C overnight, the cells were treated with NiCl₂ for 24 h. RT-PCR and Western blot were carried out as described previously. E. The graphic representation of ΔHRE cyclin D1 reporter plasmid construction. 2 × 10⁴ cyclin D1-luc plasmid transiently transfected Beas-2B cells and ΔHRE cyclin D1-luc transiently transfected Beas-2B cells (F) were seeded into each well of a 48-well plates respectively. After being cultured at 37°C overnight, the cells were treated with 0.25 mmol/L or 0.5 mmol/L NiCl₂ for 12 h. The results of relative cyclin D1 induction were analyzed as described above.