Figure 3

RUNX1 attenuates STAT3 phosphorylation after cytokine treatment. (a) Phospho-STAT3 (Y705, p-STAT3) protein expression in whole-BM cells. Densitometry ratios of p-STAT3 to total STAT3 were quantified. Whole-BM cells isolated from Runx1^+/+ and Runx1^+/− mice were serum-starved for 1 h and subjected to 100 ng/ml G-CSF stimulation for the indicated duration. Mean±s.d. of the densitometry ratio and the representative western blot from three independent experiments is shown. (b) Graphical representation of the mean fluorescence intensity (MFI) of p-STAT3 within the KSL and KL compartments as assessed by Phosflow analysis. Sorted fractions were serum-starved for 1 h and subjected to 100 ng/ml G-CSF for 1 h. Data represent mean±s.d. (n=4/genotype). Results were pooled from two independent experiments. (c) Quantitative real-time PCR for Csf3r expression in KSL cells. Expression of HPRT1 was used as endogenous control. Data represent mean±s.d. (n=3/genotype). (d) Graphical representation (left) and representative flow cytometry plots (right) of p-STAT3 in 32Dcl3 cells. RUNX1-GFP or GFP⁻ transfected 32Dcl3 cells were stimulated with 50 ng/ml G-CSF. The MFI of p-STAT3 and representative flow cytometry plots for GFP-positive transfected populations are shown. (e) Western blot analysis of p-STAT3 expression in HEK293T cells transfected with RUNX1 or control plasmid. Densitometry ratios of p-STAT3 to total STAT3 were quantified. Mean±s.d. of the densitometry ratio and the representative western blot from three independent experiments are shown. (f) Graphical representation of p-STAT3 in HEK293T cells. HEK293T cells were transfected with GFP empty vector or GFP-RUNX1 for at least 24 h before stimulation with 50 ng/ml oncostatin-M (OSM). The percentage of p-STAT3 positive cells (left) and MFI of p-STAT3 (right) were quantified. Data represent mean±s.d.. Three independent experiments were performed. Asterisks represent significant differences (*P<0.05, **P<0.01, ***P<0.001 and two-tailed Student’s t-test).