Figure 7.
RUNX2 and BHLHE40 expression confers TRM_2 signature. (A) Overview of the experimental steps. (B) PCA plot illustrating the variance in gene expression profiles across the four sample groups. Each point represents a sample, with its position reflecting the overall transcriptomic differences between groups. (C) Venn diagram showing the overlap number of upregulated genes (fold change > 2, P < 0.05). (D) Heatmap displaying the expression levels of selected genes across biological replicates for the following groups: control (NC), RUNX2 overexpression (RUNX2), BHLHE40 overexpression (BHLHE40), and RUNX2 and BHLHE40 overexpression (both). Rows represent individual genes, and columns represent samples, with color intensity indicating relative expression levels. n = 3 biological replicates. (E) Reactome pathway enrichment analysis was conducted on the differentially expressed genes identified in the RUNX2 and BHLHE40 overexpression groups. Significantly enriched pathways (FDR < 0.05) are shown in the bar chart. (F) GSEA was performed on the ranked list of genes from the differential expression analysis between RUNX2 and BHLHE40 overexpression samples and controls. The enrichment plot illustrates the distribution of genes from the Interferon Gamma Signaling and Cytokine Signaling in Immune System gene sets across the ranked gene list. The NES and FDR are provided. (G) Single-cell GSEA performed on the CITE-seq data (Fig. 1). Gene sets from the top 100 genes in RUNX2, BHLHE40, and RUNX2 and BHLHE40 overexpression were applied to the CITE-seq data. (H) GSEA of differentially expressed genes of TRM_2 in CITE-seq data performed on RUNX2, BHLHE40, and both overexpression groups. (I) FACS analysis of CD103, IFN-γ, and GZMB in RUNX2- and BHLHE40-overexpressed T cells. n = 6 per group. Statistical significance for the comparisons was determined using RM one-way ANOVA, Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001. (J) Chromatin landscape at the BHLHE40 locus as revealed by scMultiome and CUT&RUN peaks for RUNX2 and H3K27ac. Regions 1 and 2 indicate predicted RUNX2-binding peaks. Blue lines in the bottom represent the association between chromatin accessibility and gene expression. NES, normalized enrichment score.

RUNX2 and BHLHE40 expression confers T RM _2 signature. (A) Overview of the experimental steps. (B) PCA plot illustrating the variance in gene expression profiles across the four sample groups. Each point represents a sample, with its position reflecting the overall transcriptomic differences between groups. (C) Venn diagram showing the overlap number of upregulated genes (fold change > 2, P < 0.05). (D) Heatmap displaying the expression levels of selected genes across biological replicates for the following groups: control (NC), RUNX2 overexpression (RUNX2), BHLHE40 overexpression (BHLHE40), and RUNX2 and BHLHE40 overexpression (both). Rows represent individual genes, and columns represent samples, with color intensity indicating relative expression levels. n = 3 biological replicates. (E) Reactome pathway enrichment analysis was conducted on the differentially expressed genes identified in the RUNX2 and BHLHE40 overexpression groups. Significantly enriched pathways (FDR < 0.05) are shown in the bar chart. (F) GSEA was performed on the ranked list of genes from the differential expression analysis between RUNX2 and BHLHE40 overexpression samples and controls. The enrichment plot illustrates the distribution of genes from the Interferon Gamma Signaling and Cytokine Signaling in Immune System gene sets across the ranked gene list. The NES and FDR are provided. (G) Single-cell GSEA performed on the CITE-seq data (Fig. 1). Gene sets from the top 100 genes in RUNX2, BHLHE40, and RUNX2 and BHLHE40 overexpression were applied to the CITE-seq data. (H) GSEA of differentially expressed genes of TRM_2 in CITE-seq data performed on RUNX2, BHLHE40, and both overexpression groups. (I) FACS analysis of CD103, IFN-γ, and GZMB in RUNX2- and BHLHE40-overexpressed T cells. n = 6 per group. Statistical significance for the comparisons was determined using RM one-way ANOVA, Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001. (J) Chromatin landscape at the BHLHE40 locus as revealed by scMultiome and CUT&RUN peaks for RUNX2 and H3K27ac. Regions 1 and 2 indicate predicted RUNX2-binding peaks. Blue lines in the bottom represent the association between chromatin accessibility and gene expression. NES, normalized enrichment score.

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