Fig. 5

Identification of DGC-specific transcriptional regulators and a potential protein that governs macrophage recruitment by DGCs. a Heat map showing H3K27ac signals of all DGC-specific enhancers in three matched GSCs and DGCs (MGG4, MGG6, and MGG8). H3K27ac chip sequencing data were derived from NCBI Gene Expression Omnibus GSE54047. b Metaplot showing average H3K27ac signals of all DGC-specific enhancers in three matched GSCs and DGCs (MGG4, MGG6, and MGG8). H3K27ac chip sequencing data were derived from NCBI Gene Expression Omnibus GSE54047. c De novo and known motif enrichment analysis of DGC-specific enhancers defined in a, b displayed enrichment for transcriptional motifs of the TEAD transcription factor family. d GSEA analysis of YAP-related signatures upregulated in DGCs compared with GSCs. NCBI Gene Expression Omnibus GSE54791. NES: normalized enrichment score. FDR: false discovery rate. e Quantification of the TEAD luciferase reporter assay of matched pairs of GSCs and DGCs derived from MGG4 and MGG8 cells. Data are presented as the mean ± SEM of four independent experiments. *P < 0.05, ***P < 0.001. f Venn diagram showing the intersection between DGC signature genes (n = 50) and genes encoding secreted proteins from the human protein atlas (n = 1708). CCN1 was selected because it is a target gene of the YAP/TAZ-TEAD complex. g H3K27ac ChIP-sequencing enrichment plot at the CCN1 locus of three matched pairs of GSCs and DGCs (MGG4, MGG6, and MGG8). Matched pairs of GSC and DGC data were derived from NCBI Gene Expression Omnibus GSE54047. h qRT-PCR quantification of CCN1 mRNA levels in matched pairs of GSCs and DGCs derived from MGG4 and MGG8 cells. Data are presented as the mean ± SEM of two independent experiments. **P < 0.01, ***P < 0.001. i ELISA quantification of secreted CCN1 protein levels in conditioned media from paired GSCs and DGCs derived from MGG4 and MGG8 cells. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01. j DGCs express elevated CCN1, YAP and TAZ protein levels relative to GSCs. Protein levels of CCN1, YAP, TAZ and SOX2 (GSC marker) were assessed by immunoblotting in two pairs of GSCs and DGCs of patient-derived glioma cell lines (MGG4 and MGG8). GAPDH was used as a loading control. k Immunofluorescence staining of SOX2 (green) and CCN1 (red) in frozen sections of human GBM specimens counterstained with DAPI (blue). Scale bar, 50 μm. Green arrowheads, SOX2-positive GSC-like cells. Red arrowheads, SOX2-negative DGC-like cells. Middle and right panels are high magnifications of the rectangle area in the left panel. l Correlation between CCN1, SOX2, and OLIG2 in TCGA GBM (HG-U133A) dataset. Red numbers indicate the correlation R-value. Pearson’s correlation test