NG2 expression reduces the levels of the GCR NR3C1 and deregulates the downstream antiapoptotic PI3K/AKT/mTOR pathway in MLLr B-ALL cells. (A) Gene Set Enrichment Analysis (GSEA) for GC-resistance signature (Autry et al⁵⁰) in FACS-sorted primary NG2^POS and NG2^NEG MLLr B-ALL blasts. (B) Gene expression microarray data showing NR3C1 expression in FACS-sorted primary NG2^POS and NG2^NEG blasts from patients with MLLr B-ALL (n = 3). (C,D) NR3C1 expression by qPCR (left panel) and protein expression by immunoblot (right panel) in FACS-sorted primary NG2^POS and NG2^NEG blasts from patients with MLLr B-ALL (n = 3) (C) and NG2^WT and NG2^KO MLL-AF4⁺ SEM cells (D). (E) Representative immunofluorescence image of NR3C1 expression in NG2^WT and NG2^KO MLL-AF4⁺ SEM cells (right panel) and quantification of the mean fluorescence intensity (left panel). (F) Uniform manifold approximation and projection (UMAP) representation of NG2^POS and NG2^NEG cells analyzed by scRNAseq in FACS-sorted MLLr B-ALL blasts. Each point on the UMAP plot represents an individual cell. (G) GSEA for hallmark signaling pathways from Molecular Signatures Database (MSigDB). Each dot represents a specific hallmark gene set, the size corresponds to the gene ratio, and the color represents the significance of each comparison. (H) Volcano plot depicting the most differentially expressed genes between NG2^POS and NG2^NEG cells. A positive fold change corresponds to genes overexpressed in the NG2^POS population, and vice versa. Genes relevant in the PIK3/AKT/mTOR signaling pathways are indicated in purple. (I) Representation of BCL2 and BCL11A expression in NG2^POS and NG2^NEG cells. The size and the color of the circles correspond to the percentage of cells expressing the indicated gene and the average expression, respectively.