FIG. 4.

Repression of the P1 promoter by RUNX3 requires the VWPRY sequence. (A) Akata 31 stable cell lines containing vector control pMEP4, pMEP4-RUNX3, or pMEP4-ΔVWRPY under the control of the metallothionein promoter were induced by the addition of CdCl₂ at 5 μM for the times indicated. RIPA extracts were prepared, and 50 μg protein was analyzed by SDS-PAGE. Western blotting was performed, probing for RUNX3, RUNX1, and β-actin as a loading control. (B) Forty-eight-hour transient transfection assay of DG75 cells with a RUNX1c promoter luciferase reporter construct (pGL3-RUNX1P1) cotransfected with the pCEP4 empty-vector or RUNX3 expression plasmid (pCEP-RUNX3 or pCEP4-ΔVWRPY). Results are expressed as means ± standard deviations of results from three transfections. Relative luciferase units (RLU) were adjusted for transfection efficiency using a cotransfected β-galactosidase (Gal) reporter plasmid. (C) HEK293 cells were transfected with the pCEP4, RUNX3 wild-type, or ΔVWRPY expression plasmid and extracts analyzed by Western blotting, probing for RUNX3 or β-actin as a loading control.