Figure 5

RUNX1 physically interacts with STAT3 to attenuate phosphorylation of STAT3 after cytokine treatment. (a) Co-immunoprecipitation of RUNX1 with STAT3 in HEK293T cells. GFP-tagged FLAGHA-STAT3, RUNX1, or GFP-expressing plasmids were overexpressed in HEK293T. Lysates were co-immunoprecipitated using GFP beads, and western blots were probed with indicated antibodies. (b) Co-immunoprecipitation of endogenous STAT3 with RUNX1 in HEK293T cells. EGFP-tagged RUNX1, or EGFP-expressing plasmids were overexpressed in HEK293T by lipofection. Lysates were co-immunoprecipitated using GFP beads, and western blots were probed with indicated antibodies. (c) Co-immunoprecipitation of RUNX1 with STAT3 in Jurkat cells. Lysates were co-immunoprecipitated with the indicated antibodies. Western blots were probed with the indicated antibodies. Representative blots from three independent experiments are shown. (d) Schematic diagram of mutant constructs of RUNX1 and STAT3. (e and f) Co-immunoprecipitation of RUNX1 mutant constructs with STAT3 (e), and reciprocal co-immunoprecipitation of STAT3 mutant constructs with RUNX1 (f). Lysates were co-immunoprecipitated using GFP beads, and western blots were probed with indicated antibodies. (g) Attenuation of p-STAT3 by RUNX1 mutants. HEK293T were transfected with indicated RUNX1 constructs for 24 h and treated with 50 ng/ml oncostatin-M (OSM) for 30 min. The percentage of p-STAT3-positive cells was determined by Phosflow analysis. Mean±s.d. of three independent experiments is shown. (h) Interaction of FPD/MM patients-associated RUNX1 mutants with STAT3. Lysates were co-immunoprecipitated using GFP beads, and western blots were probed with indicated antibodies. (i) Attenuation of p-STAT3 by RUNX1 or FPD/MM patients-associated RUNX1 mutants. HEK293T were transfected with RUNX1, R174Q or Y260X for 24 h and treated with 50 ng/ml OSM for 30 min. The percentage of p-STAT3-positive cells was determined by flow cytometry. Mean±s.d. of three independent experiments is shown. Asterisks represent significant differences (*P<0.05, **P <0.01, ***P<0.001 and two-tailed Student’s t-test).