Moderate inhibition of RUNX1 paradoxically upregulates total RUNX expression. (A) Expressions of RUNX1, RUNX2, RUNX3, and total RUNX (Pan_RUNX) were determined in MV4-11 cells transduced with control (sh_Luc.) or with RUNX1 shRNAs (sh_Rx1_moderate and sh_Rx1_profound) in the presence of 3 μM of doxycycline for 24 h. Values are normalized to those of control vector-transduced cells (n = 3). (B) Schematic illustrations show proximal promoter region (−2000 bp to +500 bp of transcriptional start site) of GSTA2. (C) ChIP analysis in MV4-11 cells using anti-RUNX1, anti-RUNX2, or anti-RUNX3 antibodies, an isotope-matched control IgG and anti-histone H3 antibody. ChIP products were amplified by PCR to determine abundance of the indicated amplicons. R1 and R2 correspond to RUNX consensus-binding sequences in the GSTA2 promoter, as is described in panel B. PRL30 was used as a negative control. (D) Luciferase reporter assay of GSTA2 promoter. HEK293T cells were transduced with the indicated lentivirus vectors as well as with luciferase reporter plasmids and then incubated with 3 μM of doxycycline. Forty-eight hours after treatment, relative luciferase activity was determined (n = 3). Data are mean ± SEM values. *P < .05, by 2-tailed Student t test; **P < .01; ***P < .001.