Fig. 3.

Effect of PML/RARα induction on the binding of PML-RARα to the HHEX gene promoter and on the expression of VEGF-A, VEGFR-2, and HHEX at RNA and at protein level. a PML-RARα binding to the HHEX promoter as shown by ChIP experiments. Nuclear extracts derived from PR9 cells grown for 24 h either in the absence (c) or in the presence of ZnSO₄ (Zn²⁺); cells were crosslinked in vivo; after cell lysis, chromatin fragments were immunoprecipitated with anti-RARα antibody; and after DNA purification, DNA regions containing HHEX were amplified. In the figure, the HHEX PCR signal without immunoprecipitation is indicated as −A (IgG control) and, after immunoprecipitation, as +A (RARα antibody). b, c, d ZnSO₄(Zn²⁺) was added to PR9 cells and, at different time points and concentrations, cell aliquots were harvested and processed for evaluation of PML-RARα expression at protein level (b) and of VEGF-A, VEGFR-2, and HHEX expression at RNA level by RT-PCR (c) and at protein level by Western blotting (d). S26 was used for RT-PCR and β-actin for Western blotting normalization