(A) Flag-RUNX3 was coexpressed with progressively increasing amounts of Myc-K-Ras-V12 or Myc-K-Ras-17N in HEK-293 cells as indicated and the levels of RUNX3 (RX3) and K-Ras were measured by immuno-blotting with anti-Flag or anti-Myc antibody. Tubulin (Tub) was immuno-blotted with anti-tubulin antibody for loading control.
(B) Progressively increasing amounts of Myc-K-Ras-V12 were expressed in HEK-293 cells and the levels of endogenous RUNX3, p21^WAF/CIP and Bim were assessed by immuno-blotting with anti-RUNX3, anti-p21^WAF/CIP and anti-Bim antibody, respectively, and the levels of RUNX3 mRNA were monitored by Northern blotting.
(C) Serially increasing amounts of Myc-K-Ras-V12 were expressed with or without HA-p14^AFR (250 ng) in A549 lung cancer cells which lack endogenous p14^AFR. Endogenous level of RUNX3 was measured by anti-RUNX3 antibody
(D) HEK-293 cells were transiently transfected with Myc-RUNX3, Myc-K-Ras-V12 and/or RUNX3 specific siRNAs. After 48 hours transfection the cells were stained with Annexin V-FITC and Propidium Iodide. Apoptotic cells were detected by flow cytometry. The percentages of early apoptotic cells (Annexin V-FITC positive/PI negative; lower right quadrant) and late apoptotic and necrotic cells (Annexin V-FITC positive/PI positive; upper right quadrant) are shown. The results are summarized as a bar graph at the bottom. The error bars indicate the standard deviation of the results of three independent experiments.

(A) Flag-MDM2 or mutant MDM2(C438A) was coexpressed with Myc-RUNX3 and HA-Ubiquitin (HA-Ub) in HEK-293 cells as indicated. Cell lysates (500 µg for each reaction) were immuno-precipitated with anti-Myc (RUNX3) antibody and analyzed by immuno-blotting with anti-HA (Ub) antibody. WT: wild type.
(B) MKN45 cells were transfected with HA-Ub and MDM2 specific siRNA and the ubiquitination of endogenous RUNX3 was measured by IP with anti-RUNX3 antibody and IB with anti-HA (Ub) antibody.
(C) Flag-MDM2 (WT) or Flag-MDM2(C438A) (mt) was coexpressed with Myc-RUNX3, HA-Ub and HA-p14^ARF as indicated. Cell lysates (500 µg for each reaction) were immuno-precipitated with anti-Myc (RUNX3) antibody and analyzed by immuno-blotting with anti-HA (Ub) antibody.

(A) HEK-293 cells were transfected with a fixed amount of RUNX3 (200 ng), and increasing amount of MDM2 (100 ng, 250 ng and 500 ng as indicated). The effect of MDM2 on the transactivation activity of RUNX3 was measured by the luciferase reporter assay.
(B) Cells were transfected with fixed amount of RUNX3 (200 ng) and MDM2 (400 ng) and increasing amount of p14^ARF (200 ng and 400 ng as indicated). The effect of p14^ARF on the MDM2-mediated suppression of on the RUNX3 transactivation activity was measured by luciferase reporter assays.
(C) Cells were transfected with a fixed amount of RUNX3 (200 ng) and increasing amount of either wild type MDM2 (WT) or MDM2(C438A) (200 ng and 400 ng as indicated). The effect of the E3 ubiquitin ligase activity of MDM2 on RUNX3 transactivation activity was measured by the luciferase reporter assay.
(D) Cells were transfected with wild type RUNX3 or lysine mutated RUNX3 and MDM2 as indicated. Mutation of three lysine residues (K129R/K186R/K192R) by itself abolished RUNX3 transactivation activity. The effect of RUNX3 ubiquitination on its transactivation activity was measured by luciferase reporter assays using pGL3-p21 promoter-luciferase (50 ng) as reporter and pCMV-β-gal (50 ng) as internal control.

(A) Flag-MDM2 was coexpressed with HA-RUNX3 (HA-RX3) or HA-p53 in HEK-293 cells and cell lysates (500 µg) were immunoprecipitated (IP) with anti-HA antibody and immuno-blotted (IB) with anti-Flag antibody. Expression of transfected genes were detected by immuno-blotting with anti-HA or anti-Flag antibody.
(B) Cell lysate (1 mg) was obtained from MKN45 cells and immuno-precipitated with anti-RUNX3 monoclonal antibody (5G4) or IgG. The immuno-precipitate was analyzed by immuno-blotting using anti-MDM2 antibody or anti-RUNX3 antibody. Input: 50 µg of cell lysate for control.
(C) HEK-293 cells were transiently transfected with fixed amount of HA-RUNX3 (0.4 µg) or HA-p53 (0.4 µg) with an increasing amount of Flag-MDM2 (0, 1.0 and 2.0 µg). The protein expression levels were analyzed by immuno-blotting using anti-HA or Flag antibody. The intensity of each band was quantitated by densitometry and the relative ratios are indicated.
(D) MKN45 and HEK-293 cells were treated with siMDM2 and the endogenous levels of MDM2 and RUNX3 was examined by immuno-blotting using anti-MDM2 antibody or anti-RUNX3 antibody.

(A) Myc-RUNX3 and its serial deletion constructs were coexpressed with Flag-MDM2 in HEK-293 cells as indicated. Expression levels of transfected genes were measured by immuno-blotting with anti-Myc or anti-Flag antibody.
(B) Purified GST-MDM2 and His-Runt domain (His-RD; aa 64–190) were mixed with E1, E2 and HA-Ub as indicated for in vitro ubiquitination. His-RD was analyzed by SDS-gel electrophoresis followed by immuno-blotting with anti-His antibody.
(C) Wild type RUNX3 and its lysine to arginine mutants (K94R, K129R, K148R, K94/148R and KR-129,186,192) were expressed with or without MDM2 and the protein levels were analyzed by IB using anti-Myc (RUNX3) and anti-Flag (MDM2) antibodies.
(D) Myc-RUNX3 and its mutants (K94R, K129R and K148R) and HA-Ub were co-expressed with or without Flag-MDM2. RUNX3 ubiquitination was measured by IP with anti-Myc (RUNX3) antibody followed by IB with anti-HA (Ub) antibody.

Hela cells were transfected with Myc-RUNX3 and Flag-MDM2 or their mutants. The subcellular localization of the expressed proteins was detected by immuno-fluorescent staining. Each nucleus was visualized by DAPI staining.
(A) Image of cells expressing Myc-RUNX3 alone or Flag-MDM2 alone. RUNX3 and MDM2 were detected exclusively in nucleus.
(B) Image of cells co-expressing Myc-RUNX3 and Flag-MDM2. Substantial level of RUNX3 was detected in cytoplasm in cells expressing MDM2.
(C) Image of cells co-expressing Myc-RUNX3 and Flag-MDM2-339. RUNX3 was detected only in the nucleus even in the cells expressing MDM2-339.
(D) Image of cells co-expressing Myc-RUNX3-K94R and Flag-MDM2. RUNX3-K94R was detected only in the nucleus even in the cells expressing wild type MDM2.