Gating strategy of cell sorting, difference in RUNX2 expression by variants, and the effect of RUNX2 and BHLHE40. (A) Gating strategy for the identification of CD4⁺ naïve T cells. (B) Gating strategy for the identification of GFP⁺ BFP⁺ CD4⁺ CD103⁺ T_RM cells and IFN-γ–positive T cells. (C) Bar graph showing the expression levels of RUNX2 variant1 and variant2 in T cells and Saos2. n = 4 per group. Statistical significance for the comparisons was determined using a paired t test, *P < 0.05, **P < 0.01. (D) Volcano plot showing upregulated and downregulated genes in RUNX2 overexpression (left), BHLHE40 overexpression (center), and both overexpression groups (right). (E) FACS analysis of CD103 and IFN-γ in RUNX1- and RUNX3-overexpressed T cells. n = 5 per group. Statistical significance for the comparisons was determined using a paired t test and RM one-way ANOVA, Dunnett’s multiple comparison test, *P < 0.05, ***P < 0.001. (F) CUT&RUN peaks for RUNX2, BHLHE40, and H3K27ac, along with ATAC-seq peaks, are shown for T_RM_2, T_RM_1, T_EM, and naïve T cells within the IFNG and GZMB regions. Solid boxes indicate open chromatin regions in T_RM_2 that colocalize with RUNX2 and BHLHE40 peaks, as detected by MACS2. The dashed box marks IFNG promoter regions without RUNX2 and BHLHE40 binding.