Fig. 6

RUNX3 bound to the SE and activated the expression of TOX2. A Correlation between RUNX3 expression with TOX2 expression in NKTL patients from GEP dataset: GSE90784. A significant positive correlation was determined by Pearson's p value = 8.39E-09, and R = 0.64. B, C The mRNA (B) and protein (C) levels of RUNX3 and TOX2 were detected by qRT-PCR and Western blot analysis upon transfection with two different pairs of RUNX3-shRNA (RUNX2-sh1, -sh2) or the scramble shRNA (Scr) in NKYS and HANK1 cells. GAPDH was measured for data normalization (B) and β-actin was used as the loading control (C). n.s.: non-specific band produced by anti-RUNX3 antibody (A-3 clone, sc-376591) in addition to specific bands at 48, 46 kD. All these results were representative for three independent biological replicates. D Relative cell growth was measured in NKYS and HANK1 cells transduced with RUNX3-shRNAs or Scr-shRNA. For each condition, cell number was counted at day 2, 4, and 6, then converted to fold change relative to the starting number at day 0. Same number of cells were seeded at day 0 and comparison was made at indicated time points for relative fold changes of cells transduced with RUNX3-shRNA versus Scr. Three biologically independent experiments were performed (mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001. E RUNX3 binding site locates within the TOX2-SE loci. F ChIP-PCR confirmed the interaction between RUNX3 and SE region of TOX2 in NKYS and HANK1 cells. Data are expressed as fold change of RUNX3 antibody-IP vs IgG control-IP. Data are representative of 3 independent IPs. Error bars indicate SD. **p < 0.01, ***p < 0.001 by two-sample, two-tailed t-test compared with the control. G Indicated vectors were transiently transfected into 293 T cells, and luciferase activity was measured using a Dual-Luciferase system. Firefly luciferase activity was normalized to co-transfected Renilla luciferase and calculated as relative fold change to pGL4.26 empty vector. Data shown represent means ± SD of three independent experiments. **p < 0.01, compared with each RUNX3-WT group (E1, E2, and E3), respectively. WT: wild type; MUT: mutant